Method of Treating Neurological Conditions with Extract of Nerium Species or Thevetia Species

ABSTRACT

A method of treating neurological condition in a subject by administration of an extract, or fraction or sub-fraction thereof, of  Nerium  species or  Thevetia  species is provided, wherein the extract, or fraction or sub-fraction thereof, excludes oleandrin and neriifolin. Alzheimer&#39;s disease, Huntington&#39;s disease or stroke are treated by administering a therapeutically effective amount of the extract, or a fraction or sub-fraction thereof, to a subject. The extract can be present in a pharmaceutical composition.

CROSS-REFERENCE TO EARLIER FILED APPLICATIONS

The present application claims the benefit of and is a divisional ofU.S. Ser. No. 13/288,559 filed Nov. 3, 2011, which claims the benefit ofU.S. Provisional Application No. 61/415,945 filed Nov. 22, 2010, andclaims the benefit of and is a continuation-in-part of U.S. applicationSer. No. 12/987,693 filed Jan. 10, 2011, now U.S. Pat. No. 8,481,086issued Jul. 9, 2013, which claims the benefit of U.S. ProvisionalApplication No. 61/293,812 filed Jan. 11, 2010, and claims the benefitof PCT International Application No. PCT/US2011/59090 filed Nov. 3,2011, and claims the benefit of and is a continuation-in-part of PCTInternational Application No. PCT/US2011/20672 filed Jan. 10, 2011, theentire disclosures of which are hereby incorporated by reference.

FIELD OF THE INVENTION

The present invention concerns a method of treating neurologicalconditions with an extract of Nerium species or Thevetia species, orpreparations (compositions, formulations) containing them. Inparticular, the invention concerns a method for treating neurologicaldisease or disorder by administration of the extract to a subject inneed thereof. The invention also includes pharmaceutical compositionscontaining fractions or sub-fractions of the extract as well as theirmethods of use and preparation.

BACKGROUND OF THE INVENTION

Neurological diseases and disorders affect brain function. Many effortshave been made to develop curative or ameliorative therapies for thesediseases and disorders; however, no comprehensive or universallycurative therapy has been developed, even though there are numerouspharmacotherapeutic approaches that have been proven to be effectiveagainst various different diseases and disorders.

Huntington's disease (HD) is an inherited disease of the brain thataffects the nervous system. It is caused by a defective gene that ispassed from parent to child. The HD gene interferes with the manufactureof a particular protein known as ‘huntington’ which appears to becrucial for proper brain development. The classic signs of HD includeemotional, cognitive and motor disturbances. Huntington's ischaracterized by jerky involuntary movements (chorea), but sometimescauses rigidity without abnormal movements, changes in using the limbs(apraxia), loss of control of bodily functions and dementia, including aprogressive deterioration of memory, speed of thought, judgment, andlack of awareness of problems and planning. There is no known cure forHuntington's disease. Although there are a number of medications to helpcontrol symptoms associated with HD such as emotional and movementproblems, there is no treatment to stop or reverse the course of thedisease. Huntington's disease has been recognized as a disease with ageneral membrane abnormality. A significantly elevated level andactivity (10 fold increase) of Na,K-ATPase has been observed inmembranes of erythrocytes and basal ganglia of Huntington's patientscompared to that of normal (Butterfield D A, Oeswein J Q, Prunty M E,Hisle K C, Markesbery W R). Increased sodium, potassium adenosinetriphosphatase activity in erythrocyte membranes in Huntington'sdisease. Ann Neurology, 4:60-62, 1978) fibroblast membranes obtainedfrom the skin of Huntington's disease patients (Schroeder F, Goetz I E,Roberts E, Membrane anomalies in Huntington's disease fibroblasts. J.Neurochem. 43: 526-539, 1984).

Alzheimer's disease is a form of dementia—a neurodegenerative diseasethat damages the brain's intellectual functions (memory, orientation,calculation, etc.), but usually preserves its motor functions. InAlzheimer's disease, the mind gradually deteriorates, causing memoryloss, confusion, disorientation, impaired judgment and other problemsthat may affect a person's ability to perform normal daily activities.The type, severity, sequence and progression of mental changes varygreatly. There is no known cure for Alzheimer's disease and no known wayto slow its progression. For some people in the early or middle stagesof the disease, medication such as tacrine may alleviate some cognitivesymptoms. Aricept (donepezil) and Exelon (rivastigmine) are reversibleacetylcholinesterase inhibitors that are indicated for the treatment ofmild to moderate dementia of the Alzheimer's type. These drugs (calledcholinesterase inhibitors) work by increasing the brain's levels of theneurotransmitter acetylcholine, helping to restore communication betweenbrain cells. Some medications may help control behavioral symptoms suchas sleeplessness, agitation, wandering, anxiety, and depression. Thesetreatments are aimed at making the patient more comfortable. Although nomedication is known to cure Alzheimer's disease, cholinesteraseinhibitors may improve performance of daily activities, or lessenbehavioral problems. Medications for the treatment of Alzheimer'sdisease currently being tested include oestrogens, nonsteroidalanti-inflammatory agents, vitamin E, selegiline (Carbex, Eldepryl) andthe botanical product gingko biloba.

Nerium oleander is an ornamental plant widely distributed in subtropicalAsia, the southwestern United States, and the Mediterranean. Its medicaland toxicological properties have long been recognized. It has beenused, for example, in the treatment of hemorrhoids, ulcers, leprosy,snake bites, and even in the induction of abortion. Oleandrin, animportant component but not the sole component of oleander extract, is acardiac glycoside.

Extraction of glycosides from plants of Nerium species has providedpharmacologically/therapeutically active ingredients from Neriumoleander. Among these are oleandrin, neriifolin (nerifolin), and othercardiac glycoside compounds. Oleandrin extracts obtained by hot-waterextraction of Nerium oleander, sold under the trademark ANVIRZEL™,contain the concentrated form or powdered form of a hot-water extract ofNerium oleander. A Phase I trial of a hot water oleander extract (i.e.Anvirzel™) has been completed (Mekhail et al., Am. Soc. Clin. Oncol.,vol. 20, p. 82b, 2001). It was concluded that oleander extracts, whichwould provide about 57 ug oleandrin/day, can be safely administered atdoses up to 1.2 ml/m²/d. No dose limiting toxicities were found.

SUMMARY OF THE INVENTION

The invention provides a method of treating a neurological conditioncomprising administering to a subject in need thereof a compositioncontaining an extract of Nerium species or Thevetia species in aneffective amount to treat said neurological condition. The inventionprovides embodiments wherein a fraction of the extract or a sub-fractionof a fraction of the extract is used in place of the unfractionatedextract, and wherein the fraction or sub-fraction of the extractexcludes oleandrin and neriifolin.

In one aspect, the invention provides a method of treating, in a subjectin need thereof, a neurological disease or disorder with a compositioncomprising an extract of Nerium species or Thevetia species, the methodcomprising:

-   determining that the subject has a neurological disease or disorder;    and-   indicating administration to the subject a composition comprising an    extract of Nerium species or Thevetia species.

Some embodiments of the invention include those wherein: 1) the subjectis prescribed and administered a therapeutically relevant dose of thecomposition; 2) the subject is administered the composition according toa prescribed dosing regimen; 3) the extract comprises one or moretherapeutically effective agents extracted from the Nerium species orThevetia species; 4) the composition further comprises one or more othertherapeutically effective agents; 5) the extract is obtained byextraction of Nerium species or Thevetia species with hot water, coldwater, supercritical fluid, organic solvent or a combination thereof; 6)the extract excludes cardiac glycoside; 7) the extract excludes atherapeutically effective amount of cardiac glycoside; 8) the extractexcludes oleandrin; 9) the composition comprises a fraction of anextract of Nerium species or Thevetia species; 10) the compositioncomprises a fraction of an extract of Nerium species or Thevetiaspecies, wherein the fraction has been prepared by liquidchromatographic fractionation of the extract; 11) the Nerium species isNerium oleander and the Thevetia species is Thevetia neriifolia; 12) theextract excludes a neriifolin; 13) the composition comprises asub-fraction of a fraction of an extract of Nerium species or Thevetiaspecies, wherein the sub-fraction has been prepared by liquidchromatographic fractionation of a fraction of the extract, and thesub-fraction excludes oleandrin and neriifolin; 14) the extract ofNerium species or Thevetia species, if it contains cardiac glycoside,provides an improved clinical response or clinical effect whenadministered in a dosage form to a subject having neurological diseaseor disorder as compared to pure cardiac glycoside administered in anotherwise similar dosage form to the subject at the same dose of cardiacglycoside; or 15) a combination thereof.

The invention also provides a method of treating a neurologicalcondition in a subject in need thereof comprising:

-   determining whether or not the neurological condition in the subject    is Alzheimer's disease, Huntington's disease, stroke or other    neurological condition;-   indicating administration of an extract of Nerium species or    Thevetia species;-   administering an initial dose of the extract to the subject    according to a prescribed initial dosing regimen for a period of    time;-   periodically determining the adequacy of the subject's clinical    response and/or therapeutic response to treatment with the extract;    and-   if the subject's clinical response and/or therapeutic response is    adequate, then continuing treatment with extract as needed until the    desired clinical endpoint is achieved; or-   if the subject's clinical response and/or therapeutic response are    inadequate at the initial dose and initial dosing regimen, then    escalating or deescalating the dose until the desired clinical    response and/or therapeutic response in the subject is achieved.

The invention also provides a method of preventing or reducing theincidence of occurrence of a neurological condition in a population ofsubjects at risk thereof, the method comprising:

-   administering an effective dose of extract of Nerium species or    Thevetia species on a recurring basis for an extended period of time    to one or more subjects in a population of subjects at risk of    suffering from a neurological condition such as Alzheimer's disease,    Huntington's disease, stroke or other neurological condition,    thereby preventing or reducing the incidence of the neurological    condition in the population.

The invention includes embodiments wherein: a) the method furthercomprises indicating administration of the extract to the one or moresubjects; b) the method further comprises administering an effectivedose of the extract to the subject according to a prescribed dosingregimen for a period of time; c) the method further comprisesperiodically determining the adequacy of one or more subject's clinicalresponse and/or therapeutic response to treatment with the extract; d)if the subject's clinical response and/or therapeutic response isadequate, then the method further comprises continuing treatment withthe extract as needed until the desired clinical endpoint is achieved;e) if the subject's clinical response and/or therapeutic response areinadequate at the initial dose and initial dosing regimen, then themethod further comprises escalating or deescalating the dose until thedesired clinical response and/or therapeutic response in the subject isachieved; f) the extract is administered to plural subjects in apopulation; g) the recurring basis is daily, every other day, everysecond day, every third day, every fourth day, every fifth day, everysixth day, weekly, every other week, every second week, every thirdweek, monthly, bimonthly, semi-monthly, every other month every secondmonth, quarterly, every other quarter, trimesterly, seasonally,semiannually and/or annually; h) the extended period is one or moreweeks, one or more months, one or more quarters and/or one or moreyears; i) the effective dose is administered one or more times in a day;j) the method further comprises identifying a population of subjects atrisk of suffering from a neurological condition such as Alzheimer'sdisease, Huntington's disease, stroke or other neurological condition;k) the population of subjects at risk is characterized by advancing ageof the subject, familial history of the neurological condition, geneticpredisposition to occurrence of neurological condition, the presence andexpression of ApoE4 gene in the subject, female gender (twice as manywomen get Alzheimer's disease than men), cardiovascular disease (e.g.high blood pressure and high cholesterol levels), diabetes (especiallyType 2 or adult onset forms of this disease), Down's Syndrome, headinjury, low levels of formal education, smoking, excessive alcoholconsumption and/or drug abuse; 1) the extract excludes a therapeuticallyeffective amount of cardiac glycoside; m) the extract excludes cardiacglycoside; or n) a combination thereof.

The invention also provides a time-delayed method of treating stroke ina subject comprising:

-   within a delay period after a subject has suffered the stroke,    administering an initial dose of extract of Nerium species or    Thevetia species according to an initial dosing regimen;-   determining the adequacy of subject's clinical response and/or    therapeutic response to treatment with the extract; and-   if the subject's clinical response and/or therapeutic response is    adequate, then continuing treatment with extract as needed until the    desired clinical endpoint is achieved; or-   if the subject's clinical response and/or therapeutic response are    inadequate at the initial dose and initial dosing regimen, then    escalating or deescalating the dose until the desired clinical    response and/or therapeutic response in the subject is achieved.

Some embodiments of the invention include those wherein: 1) the delayperiod is 10 hours or less, 8 hours or less, 6 hours or less, 4 hours orless, 3 hours or less, 2 hours or less, 1 hour or less, 45 minutes orless, 30 minutes or less, 20 minutes or less or 10 min or less; 2)determining the adequacy of a subject's clinical and/or therapeuticresponse is done by assessments of any weakness of the face, arm and/orleg on one side of the body, numbness in the face, arm, and/or leg onone side of the body, inability to understand spoken language, inabilityto speak or speak clearly, inability to write, vertigo and/or gaitimbalance, double vision and an unusually severe headache; or 3) acombination thereof.

The invention also provides use of an extract of Nerium species orThevetia species in the manufacture of a medicament for the treatment ofa neurological condition in a subject. In some embodiments, themanufacture of such a medicament comprises: providing an extract ofNerium species or Thevetia species; including a dose of extract ofNerium species or Thevetia species, or a fraction thereof, in apharmaceutical dosage form; and packaging the pharmaceutical dosageform. The invention also provides a pharmaceutical compositioncomprising an extract of Nerium species or Thevetia species for thetreatment of a neurological condition in a subject. In some embodiments,the manufacture can be conducted as described in PCT InternationalApplication No. PCT/US06/29061 filed Jul. 26, 22006, U.S. Pat. No.7,402,325 filed Jul. 28, 2005, or U.S. Ser. No. 12/019,435 filed Jan.24, 2008, the entire disclosures of which are hereby incorporated byreference. The manufacture can also include one or more additional stepssuch as: delivering the packaged dosage form to a vendor (retailer,wholesaler and/or distributor); selling or otherwise providing thepackaged dosage form to a subject having a neurological condition;including with the medicament a label and a package insert, whichprovides instructions on use, dosing regimen, administration, contentand toxicology profile of the dosage form. In some embodiments, thetreatment of a neurological condition comprises: determining that asubject has a neurological disease or disorder; indicatingadministration of the extract, or a fraction thereof, to the subjectaccording to a dosing regimen; administering to the subject one or morepharmaceutical dosage forms containing the extract, wherein the one ormore pharmaceutical dosage forms is administered according to the dosingregimen.

The invention also provides an extract of Nerium species or Thevetiaspecies, or a composition, i.e. a pharmaceutical formulation or dosageform, comprising an extract of Nerium species or Thevetia species forthe treatment of a neurological condition. In some embodiments, theextract can be obtained from Nerium species or Thevetia species asdescribed herein or in U.S. Pat. No. 7,402,325, PCT InternationalApplication No. PCT/US06/29061, U.S. application Ser. No. 12/019,435, orNewman et al. (Mol. Interven. (2008), 8, 36-49), the entire disclosuresof which are hereby incorporated by reference.

The invention provides a method for preparing a fraction of extract ofNerium species or Thevetia species comprising: extracting a masscomprising Nerium species or Thevetia species to form an unfractionatedextract thereof, the extract comprising one or more pharmacologicallyactive components for the treatment of a neurological condition; andfractionating the unfractionated extract to form two or more fractionsthereof, wherein at least one fraction comprises one or more non-cardiacglycoside pharmacologically active components. In some embodiments, a)at least one fraction excludes cardiac glycoside; b) at least onefraction further comprises cardiac glycoside; c) the extraction isconducted with supercritical fluid, water, organic solvent or acombination thereof; d) the fractionation is conducted by liquidchromatography or solvent extraction; e) the at least one fractionexcludes oleandrin and neriifolin; or f) a combination thereof.

The invention also provides a method of fractionating an extract ofNerium species or Thevetia species in order to provide one or moretherapeutically effective fractions thereof. The method comprises: a)providing an extract of Nerium species or Thevetia species; b)fractionating the extract to provide two or more different fractions ofthe extract, a first extract fraction comprising one or morepharmacologically active agents, which is/are not a cardiac glycoside,and excluding cardiac glycoside (oleandrin and neriifolin), and a secondextract fraction comprising one or more cardiac glycosides and one ormore pharmacologically active agents, which is/are not a cardiacglycoside. The fractionation can also be performed as described herein.In some embodiments, the first or second extract fraction is subjectedto further fractionation to provide two or more different sub-fractions,wherein a first sub-fraction comprises one or more steroids and a secondsub-fraction comprises one or more triterpenes. In some embodiments, thefractionation is performed by liquid chromatography with a stationaryphase and a mobile phase.

The invention also provides a composition comprising a fraction of anextract obtained from Nerium species or Thevetia species, whereby thefraction has been obtained by fractionation of the extract obtained fromNerium species or Thevetia species. In some embodiments, a fraction ofextract comprises one or more steroids and one or more tritepenes andoptionally excludes cardiac glycoside (oleandrin and neriifolin).

The invention also provides a composition comprising a sub-fraction offraction of an extract obtained from Nerium species or Thevetia species,whereby the sub-fraction has been obtained by further fractionation of afraction of the extract obtained from Nerium species or Thevetiaspecies. In some embodiments, a sub-fraction of a fraction of extractcomprises one or more steroids, cardiac glycosides, the associatedaglycones of cardiac glycosides, e.g. oleandrigenin, cardenolides, ortriterpenoids, and one or more tritepenes. In some embodiments, asub-fraction of a fraction of extract comprises one or more triterpenesand excludes a steroid. Each sub-fraction independently optionallyexcludes cardiac glycoside (oleandrin and neriifolin).

In some embodiments: a) the extract further comprises at least twopharmacologically active agents obtained (extracted) from Nerium speciesor Thevetia species; b) the at least two pharmacologically active agentsfunction additively or synergistically to contribute to the therapeuticefficacy of the extract when the extract is administered to a subject;c) none of the at least two pharmacologically active agents is a cardiacglycoside; and/or d) at least two pharmacologically active agents areselected from the group of cardiac glycosides, the associated aglyconesof cardiac glycosides, e.g. oleandrigenin, cardenolides ortriterpenoids.

In some embodiments: 1) the cardiac glycoside is selected from the groupconsisting of oleandrin, odoroside, neritaloside, ouabain, bufalin,digitoxin, cinobufatalin, cinobufagin, and resibufogenin; 2) the extractis present in a pharmaceutical formulation or composition; 3) theextract has been obtained from an oleander plant mass or neriifoliaplant mass; 4) the plant mass comprises Nerium species, such as Neriumoleander, or Thevetia species, such as Thevetia neriifolia or Thevetiaperuviana (otherwise known as yellow oleander); 5) the extract wasprepared by supercritical fluid (SCF) extraction optionally in thepresence of a modifier; 6) the cardiac glycoside is oleandrin; 7) theextract was prepared by hot water extraction, cold water extraction,organic solvent extraction or aqueous organic solvent extraction.

In some embodiments, the extract (or fraction or sub-fraction thereof)comprises less than 1% wt., less than 0.5% wt., less than 0.1% wt., lessthan 0.05% wt. or less than 0.01% wt. of polysaccharide. In someembodiments, the extract (or fraction or sub-fraction thereof) comprisesbetulin (urs-12-ene-3β,28-diol); 28-norurs-12-en-3β-ol; urs-12-en-3β-ol;3β,3β-hydroxy-12-oleanen-28-oic acid; 3β,20α-dihydroxyurs-21-en-38-oicacid; 3β,27-dihydroxy-12-ursen-38-oic acid;3β,13β-dihydroxyurs-11-en-28-oic acid;3β,12α-dihydroxyoleanan-28,13β-olide; and3β,27-dihydroxy-12-oleanan-28-oic acid. In some embodiments, the extract(or fraction or sub-fraction thereof) comprises one or more cardiacglycoside precursors selected from a glycone constituent of a cardiacglycoside. In some embodiments, the glycone is selected from the groupconsisting of glucoside, fructoside, and glucuronide. In someembodiments, the extract (or fraction or sub-fraction thereof) comprisesoleandrigenin, ursolic acid, betulinic acid, odoroside, neritaloside,oleanolic acid and one or more triterpenes and less than 0.5% by weightpolysaccharide.

In some embodiments, the subject having a neurological condition, i.e.the subject in need thereof, is part of a population of such subjects.The invention provides a method for improving the clinical status of astatistically significant number of subjects of in a population ofsubjects having a neurological condition, the method comprising:administering to the population of subjects an extract of Nerium speciesor Thevetia species or a composition comprising an extract of Neriumspecies or Thevetia species; and determining the clinical status of thesubjects. In some embodiments, the statistically significant number isat least 5% of the population.

In some embodiments, the neurological condition is Alzheimer's disease,Huntington's disease, stroke, a tauopathy or other neurologicalcondition, such as described herein. The medicament can be manufacturedby inclusion of the extract in a pharmaceutical dosage form containingone or more pharmaceutically acceptable excipients.

Treatment of the subject with the extract or composition containing theextract is continued as needed. The dose or dosing regimen can beadjusted as needed until the patient reaches the desired clinicalendpoint(s) such as a reduction or alleviation of specific neurologicalsymptoms associated with the disease. Determination of the adequacy ofclinical response and/or therapeutic response can be conducted by aclinician familiar with the neurological condition being treated.

In some embodiments, the neurological condition is selected from thegroup consisting of neurological disease, neurological disorder,tauopathy, and stroke. In some embodiments, the neurological disease isa neurodegenerative disease. In some embodiments, the neurodegenerativedisease is selected from the group consisting of Huntington's disease,Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis,bovine spongiform encephalopathy, multiple sclerosis, diabeticneuropathy, autism and juvenile neuronal ceroid lipofuscinosis. In someembodiments, stroke is stroke-mediated ischemic injury. In someembodiments, the neurological condition is a tauopathy, which is aneurodegenerative disease having an etiology associated with animbalance in the Tau3R/Tau4R ratio in a subject. Tauopathies are a classof neurodegenerative diseases resulting from the pathologicalaggregation of tau proteins in the human brain. In some embodiments, thetauopathy is Down's syndrome, Pick's disease, corticobasal degeneration,some variants of prions disease, Alzheimer's disease, progressivesupranuclear palsy or frontotemporal dementia. The individual steps ofthe methods of the invention can be conducted at separate facilities orwithin the same facility.

In some embodiments, the neurons are in vitro, ex vivo or in vivo. Insome embodiments, the neurons are CA-1 neurons.

In some embodiments, the invention provides an extract, or fractionthereof or sub-fraction thereof, of Nerium species or Neriifoliaspecies, having a ¹HNMR spectrum as described herein. In someembodiments, the invention provides an extract, or fraction thereof orsub-fraction thereof, of Nerium species or Neriifolia species,exhibiting therapeutic activity as described herein when administered toa subject. In some embodiments, the invention provides an extract, orfraction thereof or sub-fraction thereof, of Nerium species orNeriifolia species, having a HPLC chromatogram as described herein. Insome embodiments, the methods of the invention employ an extract,fraction thereof or sub-fraction thereof, as described herein. In someembodiments, the compositions of the invention comprise an extract,fraction thereof or sub-fraction thereof, as described herein.

The invention includes all combinations of the aspects, embodiments andsub-embodiments of the invention disclosed herein. Unless otherwisespecified herein, the term “extract” can refer to the unfractionatedextract or fractionated extract, i.e. a fraction of the extract, orsub-fractionated extract, i.e. a sub-fraction of a fraction of theextract.

BRIEF DESCRIPTION OF THE FIGURES

The following figures form part of the present description and describeexemplary embodiments of the claimed invention. The skilled artisanwill, in light of these figures and the description herein, be able topractice the invention without undue experimentation.

FIG. 1A depicts concentration-response data obtained from thecomparative evaluation of the oleandrin versus no oxygen or glucosedeprivation (OGD), the control, in a neuroprotection brain-slice-based“stroke” assay (Example 8), wherein the number of healthy corticalneurons is determined following 5-6 minutes of oxygen and glucosedeprivation (OGD=stroke) in the presence or absence of oleandrin.

FIG. 1B depicts results of a concentration-response assay forunfractionated SCF extract of Nerium oleander in a neuroprotectionbrain-slice-based “stroke” assay as described herein (Example 8),wherein no oxygen or glucose deprivation is used as the control.

FIGS. 2A-2C depict results of the comparative evaluation of oleandrinversus the unfractionated SCF extract of Nerium oleander in aneuroprotection brain-slice-based “Alzheimer's” assay (Example 9),wherein the number of healthy cortical neurons is determined followingAPP/Aβ-induced degeneration in the absence or presence of varying amountof those agents.

FIGS. 3A-3D depict results from duplicate experiments of the comparativeevaluation of oleandrin (FIGS. 3A-3B) (FIGS. 3C-3D) in a neuroprotectioncortico-striatal co-culture neuron-based “Huntington's disease” assay(Example 10), wherein the percent rescue, relative to control, ofcortical neurons versus striatal neurons transfected with a mutant formof the Huntington (htt) protein is determined in the absence or presenceof varying amounts of oleandrin.

FIGS. 4A-4E depict the results of a neuroprotection brain-slice-based“stroke” assay as described herein, wherein the oleandrin-containing SCFextract has been fractionated via liquid chromatography (Example 13) andthe five different fractions (described below) subjected to this assay(Example 15): Fraction O-H (FIG. 4A), Fraction O-2 (FIG. 4B), FractionO-3 (FIG. 4C), Fraction O-4 (FIG. 4D), Fraction O-5 (FIG. 4E).

FIG. 5 depicts the results of a concentration-response brain-slice-based“stroke” assay (Example 15) for fraction O-4 of the SCF extract ofNerium oleander versus the parent unfractionated Nerium oleander SCFextract (PBI or PBI-05204).

FIG. 6 depicts the results of the comparative evaluation of a fraction(O-4 or O-4A) of Nerium oleander SCF extract versus untreated (cellswere not transfected with APP/Aβ) in an APP-based “Alzheimer's” assay(Example 11), wherein the number of healthy cortical neurons isdetermined following APP/Aβ-induced degeneration in the absence orpresence of varying amount of those agents.

FIG. 7 depicts the results of the comparative evaluation of a fraction(O-4) of Nerium oleander SCF extract versus untreated (cells were nottransfected with APP/Aβ) in a Tau4R based “Alzheimer's” assay (Example12), wherein the number of healthy and damaged cortical neurons aredetermined following Tau4R in the absence or presence of varying amountsof those agents.

FIGS. 8A-8D depict the chromatograms obtained by HPLC analysis of thefractions prepared according to Example 13.

FIGS. 9A-9I depict HNMR spectra for various components present in thefraction O-4 Nerium oleander SCF extract. FIG. 9A depicts the HNMRspectrum of the O-4 fraction before sub-fractionation according toExample 17. FIGS. 9B-9I depict the HNMR spectra for varioussub-fractions obtained by silica gel flash chromatography, according toExample 17, performed on the O-4 fraction.

DETAILED DESCRIPTION OF THE INVENTION

The invention provides a method of treating a neurological condition byadministration of an effective dose of extract of Nerium species orThevetia species to a subject in need thereof. The extract isadministered according to a dosing regimen best suited for the subject,the suitability of the dose and dosing regimen to be determinedclinically according to conventional clinical practices and clinicaltreatment endpoints for the neurological condition being treated.

In some embodiments, the neurodegenerative disorder or neurologicalcondition being treated has an etiology associated with anover-expression of tau proteins and/or an imbalance in the Tau3R/Tau4Rratio in a subject. Such a condition is termed a tauopathy. Exemplarytauopathies include Down's syndrome, Pick's disease, some variants ofprions disease, Alzheimer's disease, progressive supranuclear palsy orfrontotemporal dementia, corticobasal degeneration, Guam parkinsonismdementia complex, dementia with argyrophilic grains, Niemann-Pickdisease Type C, and dementia pugilistic.

In some embodiments, the neurodegenerative disorder or neurologicalcondition being treated has an etiology associated with abnormal oratypical proteolysis of amyloid beta precursor protein, accumulation ofamyloid beta protein in the synapses of the neurons, formation ofamyloid fibrils in the synapses of the neurons, or formation of amyloidplaques in the synapses of the neurons. Exemplary of such disorders orconditions is Alzheimer's disease. A subject treated according to theinvention will exhibit a therapeutic response. By “therapeutic response”is meant that a subject suffering from the disease or disorder willenjoy at least one of the following clinical benefits as a result oftreatment with the extract: amelioration of the disease or disorder,reduction in the occurrence of symptoms associated with the disease ordisorder, partial remission of the disease or disorder, full remissionof the disease or disorder, or increased time to progression. In otherwords, the therapeutic response can be a full or partial therapeuticresponse.

A therapeutic response can also be described as one in which the qualityof life of the patient afflicted with the neurodegenerative disease isimproved Improvement in quality of life may occur, for example, througha reduction in occurrence, frequency or severity of symptoms associatedwith the disease (e.g. tremors, involuntary muscle movements, loss orpartial loss of nerve-muscle coordination, memory retention, etc.).

“Preventing occurrence of a neurological condition in a population ofsubjects at risk” means that the neurological condition will not occurduring a predetermined time period in a demographically predeterminedpopulation of subjects that are at risk of suffering from theneurological condition. The prevention during the predetermined timeperiod occurs as a result of subjects in that population having beenadministered an extract according to the invention. As one example, whenan extract or extract-containing composition is administered for apredetermined time period to subjects in a population of subjects atrisk of suffering from stroke, stroke will not occur in those subjectsduring the predetermined time period. In particular, anextract-containing composition is chronically administered over a periodof one year to a population of subjects at risk of suffering fromAlzheimer's disease or any of the tauopathology related diseases, andthe subjects in that population do not exhibit symptoms associated withAlzheimer's during that one-year period.

“Reducing the incidence of occurrence of a neurological condition in apopulation of subjects at risk” is related in meaning to “preventing theincidence”, except that “reducing the incidence of occurrence” permitsthe occurrence of the neurological condition in a demographicallypredetermined population of subjects but at a rate of occurrence or alevel of severity that is reduced as compared to an otherwisedemographically similar predetermined population of subjects at risk notbeing administered the extract-containing composition according to theinvention.

As used herein, “time to progression” is the period, length or durationof time after a disease is diagnosed (or treated) until the diseasebegins to worsen. It is the period of time during which the level of adisease is maintained without further progression of the disease, andthe period of time ends when the disease begins to progress again.Progression of a disease is determined by “staging” a subject sufferingfrom a neurological condition prior to or at initiation of therapy. Forexample, the subject's neurological health is determined prior to or atinitiation of therapy. The subject is then treated with the extract, andthe neurological health monitored periodically. At some later point intime, the symptoms of the neurological condition may worsen, thusmarking progression of the disease and the end of the “time toprogression”. The period of time during which the disease did notprogress or during which the level or severity of the disease did notworsen is the “time to progression”.

A dosing regimen includes a therapeutically relevant dose (or effectivedose) of extract administered according to a dosing schedule. Atherapeutically relevant dose, therefore, is a therapeutic dose at whicha therapeutic response of the disease or disorder to treatment withextract is observed and at which a subject can be administered theextract without an excessive amount of unwanted or deleterious sideeffects. A therapeutically relevant dose is non-lethal to a subject,even though it may cause some side effects in the patient. It is a doseat which the level of clinical benefit to a subject being administeredthe extract exceeds the level of deleterious side effects experienced bythe subject due to administration of the extract. A therapeuticallyrelevant dose will vary from subject to subject according to a varietyof established pharmacologic, pharmacodynamic and pharmacokineticprinciples. However, a therapeutically relevant dose will typically bein the range of 0.1 to 100 micrograms of extract/day, the extract beingin either solid, liquid or semisolid form. It is known in the art thatthe actual amount of a pharmacologically active agent required toprovide a target therapeutic result in a subject may vary from subjectto subject according to the basic principles of pharmacy.

A therapeutically relevant dose can be administered according to anydosing regimen typically used in the treatment of neurological orneurodegenerative diseases or disorders. A therapeutically relevant dosecan be administered once, twice, thrice or more daily dosing schedule.It can be administered every other day, every third day, every fourthday, every fifth day, semiweekly, weekly, biweekly, every three weeks,every four weeks, monthly, bimonthly, semimonthly, every three months,every four months, semiannually, annually, or according to a combinationof any of the above to arrive at a suitable dosing schedule. Forexample, a therapeutically relevant dose can be administered once dailyfor one or more weeks.

The examples below include evidence of the efficacy of the extract inneurological conditions such as neurological diseases, neurologicaldisorders and stroke. Example 3 details a method of treating Alzheimer'sdisease with a Nerium species extract, or composition thereof, Thevetiaspecies extract, or composition thereof, or a combination thereof withone or more other therapeutic agents. Example 4 details a method oftreating Huntington's disease with the extract or a combination of theextract with one or more other therapeutic agents. Example 5 details amethod of treating stroke-mediated and non-stroke mediated ischemicbrain injury with the extract or a combination of the extract with oneor more other therapeutic agents.

In general, a subject having a neurological condition is treated asfollows. A subject presenting with a neurological condition is evaluatedto determine whether or not the neurological condition is Alzheimer'sdisease, Huntington's disease, stroke or other neurological condition.If the subject has a positive diagnosis, administration of the extractor extract-containing composition is indicated. Initial doses of theextract or composition are administered to the subject according to aprescribed dosing regimen for a period of time. The subject's clinicalresponse and level of therapeutic response are determined periodically.If the level of therapeutic response is too low at one dose, then thedose is escalated according to a predetermine dose escalation scheduleuntil the desired level of therapeutic response in the subject isachieved. If the subject exhibits undesirable side effects or anunacceptable level of side effects, then the dose is deescalated untilthe desired balance of level of therapeutic response versus side effectprofile in the subject is achieved. Treatment of the subject with theextract or composition is continued as needed. The dose or dosingregimen can be adjusted as needed until the patient reaches the desiredclinical endpoint(s) such as cessation of the disease itself, reductionin disease associated symptoms, and/or a reduction in the progression ofthe disease process.

The extract, in particular unfractionated extract, comprises one or morepharmacologically active compounds. Some of those compounds are as yetunidentified and some can be oleandrin or other cardiac glycosides,oleaside, oleandrigenin, neritaloside, odoroside (Wang X, Plomley J B,Newman R A and Cisneros A. LC/MS/MS analyses of an oleander extract forcancer treatment, Analytical Chem. 72: 3547-3552, 2000), and other plantmaterials. Unfractionated SCF extract from a supercritical fluid processtypically contains a theoretical range of 0.9% to 2.5% by weight ofoleandrin. SCF extracts comprising varying amount of oleandrin have beenobtained. In one embodiment, the SCF extract comprises about 2% by wt.of oleandrin.

The extractable unidentified components of the extract of Nerium speciesor Thevetia species can comprise at least one (non-cardiac glycoside)pharmacologically active component that contributes to the efficacy ofthe SCF extract or a fraction thereof. Two or more pharmacologicallyactive extractable components can function additively or synergisticallyto provide the observed efficacy. In other words, the Nerium species orThevetia species extract of the invention comprises one or morepharmacologically active components that are not a cardiac glycoside,even though one or more cardiac glycosides can additionally be includedin the extract. The extract can be fractionated into various differentfractions some of which contain cardiac glycoside, one or morenon-cardiac glycoside pharmacologically active components or acombination thereof. In addition, each fraction of extract can befurther fractionated into two or more different sub-fractions.

Evidence of the existence of one or more pharmacologically activecomponents, other than oleandrin, in the SCF extract was obtained bycomparing the concentration-response curves for a solution containingpure oleandrin versus one containing the SCF extract. FIG. 1A depictsthe results of a concentration-response assay for a solution containingpure oleandrin in a neuroprotection brain-slice-based “stroke” assay asdescribed in Example 8. The concentration of oleandrin in the solutionwas varied from 0.0069 to 230 μg/ml. FIG. 1B depicts results of aconcentration-response assay for an oleandrin-containing SCF Neriumspecies extract in a neuroprotection brain-slice-based “stroke” assay asdescribed herein (Example 8). The data demonstrate that the extract ismore efficacious that pure oleandrin meaning the extract contains one ormore pharmacologically active agents that provide neuroprotection.

Example 8 provides a detailed description of an in vitro assay used toevaluate the efficacy of the extract, or composition thereof, for thetreatment of stroke-mediated ischemic neuronal injury. The assay is abrain slice-based assay for oxygen and glucose deprivation (OGD) used toinduce ≧50% loss of healthy cortical neurons by 24 hours. The parentunfractionated SCF extract of Nerium species, e.g. Nerium oleander, isused as a positive control. The parent extract is then fractionatedaccording to Example 13 to provide a fraction of extract of Neriumspecies. The fractions are analyzed according to Examples 6, 14 and 17.

The HNMR of triterpene is characterized by 7 methyl signals at upfield,an olefinic proton at ca. 5.3 ppm, and an oxygenated methine signal atca 3.4 ppm along with many methylene and methine proton signals atupfield (ca. 1.0˜2.5 ppm). The HNMR spectra (FIGS. 9B-9I) indicated themajor components as steroids and triterpenes. No signals for significantquantity of glycosides were observed. No signals for α,β-unsaturated γ-or δ-lactones, which are characteristic for cardiac glycosides, wereobserved, suggesting that there is no cardiac glycoside or its aglyconeexisting in the Fr-O-4 fraction. The HNMR spectrum in FIG. 9Ccorresponds to a sub-fraction comprising at least one steroid and atleast one or at least two different triterpene. The HNMR spectrum inFIG. 9B corresponds to a sub-fraction comprising at least two differenttripenes, such as a mixture of two ursanes, and excluding a steroid.

Accordingly, the fraction O-4 comprises at least one triterpene and atleast one steroid. In some embodiments, the fraction O-4 comprises atleast two different triterpenes and at least two different steroids, orthe fraction comprises plural different triterpenes and plural differentsteroids. The O-4 fraction evaluated in this example excludes atherapeutically effective amount of cardiac glycoside. In someembodiments, the fraction O-4 excludes a cardiac glycoside. In someembodiments, a first sub-fraction of the fraction O-4 comprises at leastone steroid and at least one triterpene or at least two differenttriterpenes, a second sub-fraction comprises at least two differenttripenes and excludes a steroid. In some embodiments, each of the firstand second sub-fractions excludes cardiac glycoside.

The fraction O-4 was tested in OGD treated brain slices (stroke model)and non-OGD treated (i.e. control) brain slices (non-stroke model). Thedata indicate that the extract O-4 fraction provides substantialneuroprotection when using solutions of extract O-4 fraction ranging inconcentration from 100 ng/mL to 1 μg/ml and provides even greaterneuroprotection when using solutions of extract O-4 fraction ranging inconcentration from 1 μg/mL to 1 mg/mL. Accordingly, a liquid dosage formcontaining 100 ng/mL to 1 mg/mL of a fraction of extract per mL ofliquid dosage form should provide neuroprotection in a subject to whichit is administered.

While no direct measurements have been made in human brain following asystemic dose of the extract, it is assumed that one or morepharmacologically active components in the fraction of extract willcross the blood brain barrier when administered to a subject. Oleandrinas pure compound or contained within the SCF extract known as PBI-05204has been shown in a rodent (mice) model to effectively cross the bloodbrain barrier and enter into the brain. It is reasonable to expect thatoleandrin would do the same with regard to a human blood brain bather.

Accordingly, the invention provides a method of protecting neuronsagainst loss of activity caused by oxygen depletion or oxygen-glucosedepletion by exposing the oxygen depleted and/or glucose-depletedneurons to an effective amount of Nerium species or Thevetia speciesextract to minimize loss of activity, reduce the rate of loss ofactivity, stop the loss of activity, slow down onset of loss ofactivity, and/or protect the function of neurons caused by exposing theoxygen depleted and/or glucose-depleted conditions. In some embodiments,the method employs an effective amount of a fraction or sub-fraction ofNerium species extract or Thevetia species extract. In some embodiments,the fraction or sub-fraction has been prepared by liquid chromatographyfractionation of the extract. In some embodiments, the fraction excludesa cardiac glycoside, and in other embodiments, the fraction orsub-fraction includes one or more cardiac glycosides, in particular ofthose described herein.

Example 9 provides a detailed description of an in vitro assay used toevaluate the efficacy of the extract for the treatment of Alzheimer'sdisease. The assay is a brain slice-based assay for APP/Aβ-induced (APP:amyloid precursor protein) degeneration of cortical pyramidal neurons.Upon cleavage by a secretase enzyme, the APP is reduced to Aβ peptideswhich are believed to be a causative factor in beta-amyloid plaqueformation. Aβ proteins are associated with beta-amyloid plaque formationand are believed to be a hallmark if not etiologic factor in Alzheimer'sdisease. Biolistic transfection is used to introduce vital markers suchas YFP (a marker yellow fluorescent protein) and to introduce diseasegene constructs into the same neuronal populations in the brain slices.YFP is co-transfected with APP isoforms leading to the progressivedegeneration of cortical pyramidal neurons over the course of three tofour days after brain slice preparation and transfection. The data(FIGS. 2A-2C) indicate that the Nerium species SCF extract provided aconcentration-dependent neuroprotection to APP-transfected brain slicesthereby rescuing levels nearly to the same levels as provided by BACEinhibitor drugs, i.e. beta secretase inhibtor drugs. The beta secretaseenzyme cleaves the APP precursor protein into toxic Aβ-proteins. Theoleandrin-containing SCF extract appeared to provide greaterneuroprotection than oleandrin alone. The data in FIGS. 2A-2C are ofsignificance in that few compounds or therapeutic strategies in theliterature have shown any significant protection of neurons in this invitro assay representative of Alzheimer disease.

The APP-WT brain slice-based Alzheimer's assay was repeated (Example 11)using fractions of the SCF extract of Nerium oleander. The number ofhealthy cortical neurons was determined following APP/Aβ-induceddegeneration in the presence of varying amounts of fraction O-4A of theSCF extract (0.01 to 100 μg/ml). Exposure to oxygen and glucosedeprivation served as the internal positive control producing thestroke-like mediated injury to neurons. The negative control was simplythe relative health of the brain slice neurons without OGD treatment orexposure to treatments. The data is depicted in FIG. 6, wherein thelighter colored bars indicate a significant difference with respect tothe APP-WT condition by ANOVA followed by Dunnett's post hoc comparisontest at the 0.05 confidence level. The data indicate that the O-4Afraction provides neuroprotection in this assay, even though it does notcontain any cardiac glycosides.

Fraction O-4 (O-4A) of the SCF extract of Thevetia oleander wasevaluated with the tau4R brain slice-based Alzheimer's assay (Example12). The number of healthy cortical neurons is determined Efficacy inthis assay is defined as or based upon the relative total number ofhealthy versus unhealthy number and percentage of degraded neurons inthe presence of varying amounts of fraction O-4A of the SCF extract (0.3to 100 μg/ml, the concentration having been determined by weight of theextract). The negative control in these experiments consisted of brainslices that were not exposed to OGD while brain slices exposed to OGDbut not treated with fractions derived from unfractionated Neriumoleander extract served as the internal positive control. The data isdepicted in FIG. 7, wherein the lighter colored bars indicate asignificant difference with respect to damaged neurons by ANOVA followedby Dunnett's post hoc comparison test at the 0.05 confidence level. Thedata indicate that the O-4A fraction provides neuroprotection in thisassay, even though it does not contain cardiac glycoside.

Accordingly, the invention provides a method of protecting neuronsagainst loss of activity caused by Alzheimer's disease, the methodcomprising: exposing the neurons exhibiting characteristics ofAlzheimer's disease to an effective amount of extract of Nerium speciesor of Thevetia species to minimize loss of activity, reduce the rate ofloss of activity, stop the loss of activity, slow down onset of loss ofactivity, and/or critical functioning of the neurons caused byAlzheimer's disease. In some embodiments, the method employs aneffective amount of a fraction of Nerium species extract or Thevetiaspecies extract. In some embodiments, the fraction has been prepared byliquid chromatography fractionation of the extract. In some embodiments,the fraction excludes a cardiac glycoside, and in other embodiments, thefraction includes one or more cardiac glycosides, in particular of thosedescribed herein.

Example 10 provides a detailed description of an assay used to evaluatethe efficacy of the extract for the treatment of Huntington's disease.Mutant htt protein is introduced via electroporation into high-density,mixed co-cultures of cortical neurons, striatal neurons, and glia. Thestriatal and cortical neurons are transfected with different colorfluorescent proteins thereby facilitating the separate identification ofthe different types of neurons in the co-culture. The color fluorescentproteins are fluorescent and ‘emit’ color upon activation with a lightsource of appropriate wavelength. The data (FIGS. 3A-3D) indicate thatoleandrin and the SCF extract of Nerium oleander are more effective thanKW6002 (an adenosine 2a receptor antagonist) in terms of providing agreater number of surviving neurons. The data also indicate that the SCFextract is more effective than oleandrin alone, suggesting that theextract further comprises one or more therapeutically effective agents,aside from oleandrin, that can be used to treat Huntington's disease.Such other agents can be used along with or in the absence of oleandrinor other cardiac glycoside. Accordingly, the invention provides a methodof protecting neurons against loss of activity caused by Huntington'sdisease, the method comprising: exposing the neurons exhibitingcharacteristics of Huntington's disease to an effective amount ofoleandrin or oleandrin-containing extract to minimize loss of activity,reduce the rate of loss of activity, stop the loss of activity, slowdown onset of loss of activity, and/or normal function of the neuronscaused by Huntington's disease.

Example 16 details an exemplary brain-slice assay that can be used toevaluate the efficacy of extract in the treatment of stroke in a subjectfollowing completion of a delay period after the stroke. The brain-sliceassay with oxygen glucose deprivation is conducted as described herein;however, rather than treating the brain slices prophylactically with theextract, they were treated with the extract after delay periods of 0, 1,2, 4, and 6 hours. The data should demonstrate that the extractcontaining is effective at providing significant neuroprotection fordelay periods of up to 1, up to 2, up to 3, up to 4, up to 5, up toabout 6 hours after the stroke.

Accordingly, the invention provides a time-delayed method of treatingstroke in a subject by administration of a dose of extract of Neriumspecies or of Thevetia species to a subject after the subject hassuffered a stroke. Within an acceptable delay period after a subject hassuffered the stroke, an initial dose of the extract is administeredaccording to an initial dosing regimen. Then, adequacy of the subject'sclinical response and/or therapeutic response to treatment with theextract is determined. If the subject's clinical response and/ortherapeutic response is adequate, then treatment with the extract iscontinued as needed until the desired clinical endpoint is achieved.Alternatively, if the subject's clinical response and/or therapeuticresponse are inadequate at the initial dose and initial dosing regimen,the dose is escalated or deescalated until the desired clinical responseand/or therapeutic response in the subject is achieved. Dose escalationor de-escalation can be performed in conjunction with a change in thedosing regimen, such as a change in dosing frequency or overall periodof dose administration.

Some of the brain slice assays herein are conducted under conditionswherein the brain tissue is treated with the extract prior to OGD. Underthose conditions, the data establishes the utility of the extract atprophylactically providing neuroprotection against damage caused bystroke.

If a clinician intends to treat a subject having a neurologicalcondition with a combination of extract, or composition thereof, and oneor more other therapeutic agents, and it is known that the particularneurological condition, which the subject has, is at least partiallytherapeutically responsive to treatment with said one or more othertherapeutic agents, then the present method invention comprises:administering to the subject in need thereof a therapeutically relevantdose of extract (or a fraction or sub-fraction thereof) and atherapeutically relevant dose of said one or more other therapeuticagents, wherein the extract (or a fraction or sub-fraction thereof) isadministered according to a first dosing regimen and the one or moreother therapeutic agents is administered according to a second dosingregimen. In some embodiments, the first and second dosing regimens arethe same. In some embodiments, the first and second dosing regimens aredifferent.

If the neurological condition being treated is Alzheimer's disease, theone or more other therapeutic agents can be selected from the groupconsisting of BACE inhibitors or acetylcholinesterase inhibitors. Insome embodiments, the one or more other therapeutic agents can beselected from the group consisting of Namenda™ (memantine HCl), Aricept™(donepezil), Razadyne™ (galantamine), Exelon™ (rivastigmine), andCognex™ (tacrine).

If the neurological condition being treated is Huntington's disease, theone or more other therapeutic agents can be selected from the groupconsisting of natural products, anticonvulsants, NMDA (n-methyld-aspartate) receptor antagonists, and sodium channel blockers.Exemplary agents include Vitamin E, Baclofen (a derivative of CoQ10),Lamotrigine (an anticonvulsant), remacemide (an anesthetic which is alow affinity NMDA antagonist), and riluzole (Na channel blocker). Theefficacy of each of these agents is considered to be low (Mestre T. etal, Chochrane Database Systematic Reviews Jul. 8, 2009; 8(3): CD006455)on its own; however, it is expected that administration of a dosage formcontaining extract to subjects receiving one or more of these otheragents will provide a subject, having a neurological disorder, animproved clinical affect as compared to administration of these agentsabsent the extract.

If the neurological condition being treated is stroke-mediated ischemicbrain injury (ischemic stroke), then the therapeutic treatmentsdisclosed in the literature (Gutierrez M. et al. “Cerebral protection,brain repair, plasticity and cell therapy in ischemic stroke”Cerebrovasc. Dis. 2009; 27 Suppl 1:177-186), e.g. intravenousthrombolysis, can be employed in addition to the extract. In someembodiments, the one or more other therapeutic agents can be selectedfrom the group consisting of drugs such as Alteplase (a thrombolyticagent).

The one or more other therapeutic agents can be administered at dosesand according to dosing regimens that are clinician-recognized as beingtherapeutically effective or at doses that are clinician-recognized asbeing sub-therapeutically effective. The clinical benefit and/ortherapeutic effect provided by administration of a combination of theextract and one or more other therapeutic can be additive orsynergistic, such level of benefit or effect being determined bycomparison of administration of the combination to administration of theindividual extract and one or more other therapeutic agents. The one ormore other therapeutic agents can be administered at doses and accordingto dosing regimens as suggested or described by the U.S. Food and DrugAdministration (U.S.F.D.A.), World Health Organization (W.H.O), EuropeanMedicines Agency (E.M.E.A.), Therapeutic Goods Administration (TGA,Australia), Pan American Health Organization (PAHO), Medicines andMedical Devices Safety Authority (Medsafe, New Zealand) or the variousMinistries of Health worldwide.

If a cardiac glycoside is used according to the invention, it can be anycardiac glycoside known to possess Na,K-ATPase binding activity. Thecardiac glycoside should be capable of crossing the blood-brain barrierand being retained in brain tissue for an extended period of timefollowing administration. In this regard, the cardiac glycoside shouldbe retained in the brain for at least 8 hours following administrationof the cardiac glycoside due to tissue binding and a consequent lowclearance rate.

If present, the cardiac glycoside can be present in pure form or as amixture with one or more other compounds. The cardiac glycoside can bepresent as an extract.

The extract can be prepared by supercritical fluid (SCF) carbon dioxide(CO₂) extraction or a chemically modified form of such an extract (e.g.an extract that includes ethanol or was made using SCF CO₂ and ethanol;Example 1). The extract can be obtained by extraction of plant materialwith an organic solvent, e.g. ethanol, methanol, propanol or other suchsolvents. The extract can be obtained from plant material. The plantmaterial can be plant mass such as obtained from Nerium species, such asNerium oleander, or of Thevetia species, such as Thevetia neriifolia orThevetia peruviana (otherwise known as yellow oleander). The extractionprocess can be conducted on a dried powder of Nerium oleander leavesprepared according to a process described in a currently-pending U.S.provisional application Ser. No. 60/653,210 filed Feb. 15, 2005 in thename of Addington or U.S. application Ser. No. 11/340,016 filed Jan. 26,2006 in the name of Addington, U.S. application Ser. No. 11/191,650filed Jul. 28, 2006 (now U.S. Pat. No. 7,402,325 issued Jul. 22, 2008)in the name of Addington, or PCT International Patent Application No.PCT/US06/29061 filed Jul. 26, 2006, or Newman et al. (Mol. Interven.(2008), 8, 36-49), the entire disclosures of which are herebyincorporated by reference, or by a process described herein. Thesemethods can also be used to prepare the unfractionated extract of Neriumspecies or of Thevetia species. Unless otherwise specified, the term“extract” as used herein can be taken to mean the “unfractionatedextract” or a fraction of the extract or a sub-fraction of a fraction ofthe extract. The term “unfractionated extract” is generally taken tomean an extract obtained by extraction of plant material, wherein theextract has not been subjected to fractionation, such as fractionationor separation into individual components or groups of components bychromatography or solvent extraction, following initial preparation ofthe extract.

As used herein, the term “oleandrin” is taken to mean all known forms ofoleandrin unless otherwise specified. Oleandrin can be present inracemic, optically pure or optically enriched form. Nerium oleanderplant material can be obtained, for example, from commercial plantsuppliers such as Aldridge Nursery, Atascosa, Tex.

The unfractionated extract can be obtained by modified (e.g. ethanol) orunmodified supercritical fluid extraction of a cardiacglycoside-containing plant mass, e.g. of a Nerium species or Thevetiaspecies containing plant mass. The supercritical fluid extract cancomprise one or more pharmacologically active agents, extracted from theplant mass, that contributes to the therapeutic efficacy of the extractwhen administered to a subject. When two or more such agents arepresent, they can contribute additively or synergistically to thetherapeutic efficacy of the extract.

The unfractionated extract can be prepared by various differentprocesses. The extract can be prepared as above or according to theprocess developed by Dr. Huseyin Ziya Ozel (U.S. Pat. No. 5,135,745)describes a hot-water extraction procedure for the preparation of theextract of the plant in water. The aqueous extract reportedly containsseveral polysaccharides with molecular weights varying from 2 KD to 30KD, oleandrin and oleandrigenin, odoroside and neritaloside. Thepolysaccharides reportedly include acidic homopolygalacturonans orarabinogalaturonans. U.S. Pat. No. 5,869,060 to Selvaraj et al.discloses hot water extracts of Nerium species and methods of productionthereof, e.g. Example 2. The resultant extract can then be lyophilizedto produce a powder. U.S. Pat. No. 6,565,897 (U.S. Pregrant PublicationNo. 20020114852 and PCT International Publication No. WO 2000/016793 toSelvaraj et al.) discloses a hot-water extraction process for thepreparation of a substantially sterile extract. Erdemoglu et al. (J.Ethnopharmacol. (2003) November 89(1), 123-129) discloses results forthe comparison of aqueous and ethanolic extracts of plants, includingNerium oleander, based upon their anti-nociceptive and anti-inflammatoryactivities. Organic solvent extracts of Nerium oleander are disclosed byAdome et al. (Afr. Health Sci. (2003) August 3(2), 77-86; ethanolicextract), el-Shazly et al. (J. Egypt Soc. Parasitol. (1996), August26(2), 461-473; ethanolic extract), Begum et al. (Phytochemistry (1999)February 50(3), 435-438; methanolic extract), Zia et al. (J.Ethnolpharmacol. (1995) November 49(1), 33-39; methanolic extract), andVlasenko et al. (Farmatsiia. (1972) September-October 21(5), 46-47;alcoholic extract). U.S. Pregrant Patent Application Publication No.20040247660 to Singh et al. discloses the preparation of a proteinstabilized liposomal formulation of oleandrin for use in the treatmentof cancer. U.S. Pregrant Patent Application Publication No. 20050026849to Singh et al. discloses a water soluble formulation of oleandrincontaining a cyclodextrin. U.S. Pregrant Patent Application PublicationNo. 20040082521 to Singh et al. discloses the preparation of proteinstabilized nanoparticle formulations of oleandrin from the hot-waterextract.

The SCF extraction can be conducted in the presence of a modifier in thesupercritical fluid, such as alcohol, e.g. ethanol, to enhanceextraction of the desired compound(s) from the plant mass(PCT/US06/29061 filed Jul. 26, 2005; U.S. Pat. No. 7,402,325; and U.S.Ser. No. 12/019,435 filed Jan. 24, 2008, or Newman et al. (Mol.Interven. (2008), 8, 36-49), the entire disclosures of which are herebyincorporated by reference). Modifiers generally possess volatilitybetween that of the supercritical fluid and of the compound beingextracted, and they must be miscible with the supercritical fluid. Insome embodiments, the modifier is a liquid at ambient conditions. By wayof example and without limitation, a modifier can be selected from thegroup consisting of ethanol, methanol, propanol, acetone, ethyl acetate,methylene chloride, etc.

It is possible that the extracts also differ in their relativeperformance as determined by efficacy in the assays included herein.Even so, if the one or more pharmacologically active agents is presentin a sufficiently high amount or concentration in the extract to be ableto prepare a therapeutically relevant dose, then the extract isconsidered part of the invention.

Example 13 describes a chromatographic method for fractionating an SCFextract into five different fractions: O-H, O-2, O-3, O-4 and O-5. Thefractions were prepared by loading the unfractionated extract onto anODS-silica gel column equilibrated with water and subsequently elutingdifferent fractions of the extract by sequentially passing variousportions of aqueous mobile phase varying in methanol content (30%, 55%,80% and 100%) through the column, collecting the respective effluents(fractions) and concentrating the effluents by solvent evaporation underreduced pressure to remove the solvent, thereby providing the fractionsO-1 (or O-H), O-2, O-3, O-4 and O-5. The fractions were analyzedaccording to Example 14 and their composition in terms of cardiacglycoside and other components was determined by thin layerchromatography using a sensitive dye indicator that adheres to (andhence is useful for detecting) cardiac glycosides. In addition, thepresence or absence of cardiac glycosides in these fractions wasanalyzed using liquid chromatography/tandem mass spectrometry or DAD-UVdetection.

A fraction or sub-fraction of the extract can be analyzed by liquidchromatography employing a stationary phase different than ODS-silicagel and/or by employing a mobile phase different than water. Exemplarysuitable stationary phases are further described herein.

FIGS. 8A-8D depict the chromatograms obtained following HPLC analysis ofthe fractions Fr-O-1, Fr-O-2, Fr-03 and Fr-O-4 of Example 13. Based upona comparison of retention times obtained using corresponding externalreference samples, it was determined the (Fr-O-2 and Fr-O-3) fractionscontain oleandrin derivatives (cardiac glycosides), oleandrin (Rt=8.3min) and other unidentified components. The bulk of the oleandrin foundin the original unfractionated SCF extract was mainly in the Fr-O-3fraction. The Fr-O-4 contained no quantifiable amounts of any cardiacglycoside. Accordingly, the composition of the fractions differedaccording to the content of oleandrin, cardiac glycoside and otherunidentified components.

Oleandrin Other Cardiac Glycoside Neuroprotection Fraction (Y/N) (Y/N)(Y/N) O-H N N Y O-2 N Y N O-3 Y Y Y O-4 (O-4A) N N Y O-5 N N N

These fractions were then subjected to the neuroprotection brainslice-based assay detailed in Example 15 to determine the level ofneuroprotection provided by each. The data are depicted in FIGS. 4A-4E,wherein the neuroprotective activity of an aqueous solution containingSCF extract (23 μg/ml) was compared to that of other solutionscontaining 0.03, 0.3 or 3 μg/ml of other component(s). All the fractionswere weighed out and compared on an equal mass weight basis. It wasdetermined that fractions (described herein) containing oleandrin, orcardiac glycoside, as well as some fractions not containing oleandrin,or cardiac glycoside, could provide neuroprotection.

Performance of fraction O-4 of the SCF extract in the brain sliced-basedstroke assay (Example 15) was compared to that of the unfractionated SCFextract (PBI-05204). The performance of varying amounts (0.03 to 300μg/ml) of the O-4 fraction was compared to a fixed amount (23 μg ofoleandrin ml) of extract. The data (FIG. 5) clearly indicates thatfraction O-4 of the SCF extract of Nerium oleander retains its efficacyeven though it does not contain oleandrin or detectable amount of anyother cardiac glycoside. The lighter colored bars in FIG. 5 indicatesignificant difference with respect to the stroke condition (set to 0)by ANOVA followed by Dunnett's post hoc comparison test at the 0.05confidence level.

Accordingly, the invention provides plural therapeutic fractions ofNerium species or Thevetia species extract, the fractions being selectedfrom the group consisting of: a) a fraction comprising one or morepharmacologically active agents and excluding oleandrin and othercardiac glycosides, wherein the fraction provides neuroprotection; b) afraction comprising one or more pharmacologically active agents,oleandrin and one or more other cardiac glycosides, wherein the fractionprovides neuroprotection; and c) a different fraction comprising one ormore other pharmacologically active agents (different than those in a)above) and excluding oleandrin and other cardiac glycosides, wherein thefraction provides neuroprotection.

The invention also provides a method of fractionating an extract ofNerium species or Thevetia species in order to provide one or moretherapeutically effective fractions thereof. The method comprises: a)providing an extract of Nerium species or Thevetia species; b)fractionating the extract to provide two or more different fractions ofthe extract, a first extract comprising one or more pharmacologicallyactive agents, which is not a cardiac glycoside, and excluding cardiacglycoside, and a second extract comprising one or more pharmacologicallyactive agents, which is not a cardiac glycoside, and one or more cardiacglycosides. In some embodiments, the fractionation is performed byliquid chromatography with a stationary phase and a mobile phase. Insome embodiments, the stationary phase comprises a medium selected fromthe group consisting of “reverse phase” resin, an inert non-polarsubstance that achieves sufficient packing for use in chromatography,e.g. composed of short (C8 to C18) carbon chains bonded to silica,cyano-bonded silica or phenyl bonded silica, ion-exchange resins (cationor anion based), “normal phase” resin, e.g. silica or organic moietieswith cyano and amino functional groups. In some embodiments, the mobilephase comprises a solvent selected from the group consisting of water,methanol, ethanol, acetonitrile, tetrahydrofuran, water based bufferedsolutions or mixtures thereof. In some embodiments, the mobile phasecomprises aqueous methanol, wherein the content of methanol is increasedsequentially from about 30% up to 100% and the stationary phase isODS-silica gel. The chromatography can be conducted using gradientelution mobile phase, stepwise elution mobile phase or a fixedcomposition mobile phase.

A fraction of extract can be sub-fractionated to provide two or moredifferent sub-fractions of a fraction of extract. Sub-fractionation canbe carried out by liquid chromatography of the fraction. A suitablestationary phase for liquid chromatography can comprise silica gel orother resins such as ion-exchange media, alumina or nonbonded C18material and a suitable mobile phase for liquid chromatography cancomprise a combination of two or more organic solvents differing inpolarity: a less polar organic solvent and a more polar organic solvent.A suitable polar organic solvent can be tetrahydrofuran,dichloromethane, ethyl acetate, acetone, dimethylformamide,acetonitrile, n-butanol, isopropanol, n-propanol, ethanol, methanol,acetic acid and water. A suitable non-polar organic solvent can be ethylacetate pentane, cyclopentane, hexane, cyclohexane, benzene, toluene,1,4-dioxane, chloroform or diethyl ether.

Buffering agents for use in buffered solutions include any of thosealready known in the art of liquid chromatography. Exemplary bufferingagents include those containing phosphate, acetate, citrate, formate,phosphate, trifluoroacetic acid, chloroacetate, sulfonate, alkyl amine,TAE, TBE, ammonia, BuffAR, carbonate, HEPES, MES, thiocyanate, CAPS,CHES, guanidine, MOPS, PIPES, TRIS, sulfate, hydroxide, alkali metalhalide, tricine, or amino acid ions or combinations thereof. One or moreion-pairing agents and/or one or more organic modifiers can also beincluded in the mobile phase.

Other types of chromatography that can be used to fractionate theextract include size exclusion chromatography, normal phasechromatography, ion exchange chromatography, hydrophobic interactionchromatography or combinations thereof. It is also possible to usecombined forms of different types of chromatography. A stationary phasecan include a medium that is a combination of two or more differentmedia used for reverse phase, size exclusion, ion exchange orhydrophobic interaction chromatography, e.g. a combination of reversephase stationary phase and size exclusion stationary phase, combinationof reverse phase stationary phase and ion exchange stationary phase, orother such combinations or two, three or four different stationary phasemedia. The stationary phase medium can be porous, non-porous, surfaceporous, diffusive porous or totally porous.

The invention provides a method of fractionating an extract comprising:a) providing an extract of extract obtained from Nerium species orThevetia species; b) fractionating the extract by column chromatography,with ODS-silica gel as stationary phase and aqueous methanol as mobilephase, to provide at least two different fractions: a first fractioncomprising at least one cardiac glycoside and at least one non-cardiacglycoside pharmacologically active agent, and another fraction excludingcardiac glycoside and comprising at least one non-cardiac glycosidepharmacologically active agent; c1) sub-fractionating the other fractionof b) by column chromatography, with silica gel as stationary phase anda mixture of at least two organic solvents differing in polarity asmobile phase, to provide at least two different sub-fractions: asub-fraction comprising one or more steroids and one or more tritepenes,and another sub-fraction comprising two or more different tripenes andexcluding a steroid, wherein the sub-fractions exclude cardiacglycoside.

In some embodiments, the method further comprises: c2) sub-fractionatingthe first fraction of b) by column chromatography, with silica gel asstationary phase and a mixture of at least two organic solventsdiffering in polarity as mobile phase, to provide at least two differentsub-fractions: a sub-fraction comprising one or more steroids and one ormore tritepenes, and another sub-fraction comprising two or moredifferent tripenes and excluding a steroid, wherein either one or bothof the sub-fractions further comprises cardiac glycoside.

The extract, or fraction or sub-fraction thereof, can be formulated inany suitable pharmaceutically acceptable dosage form. Parenteral, otic,ophthalmic, nasal, inhalable, buccal, sublingual, enteral, topical,oral, peroral, and injectable dosage forms are particularly useful.Particular dosage forms include a solid or liquid dosage forms.Exemplary suitable dosage forms include tablet, capsule, pill, caplet,troche, sache, solution, suspension, dispersion, vial, bag, bottle,injectable liquid, i.v. (intravenous), i.m. (intramuscular) or i.p.(intraperitoneal) administrable liquid and other such dosage forms knownto the artisan of ordinary skill in the pharmaceutical sciences.

The amount of extract, or fraction or sub-fraction thereof, incorporatedin a dose of the invention will be at least one or more dosage forms andcan be selected according to known principles of pharmacy. An effectiveamount or therapeutically relevant amount of therapeutic compound isspecifically contemplated. By the term “effective amount”, it isunderstood that, with respect to, for example, pharmaceuticals, apharmaceutically effective amount is contemplated. A pharmaceuticallyeffective amount is the amount or quantity of active ingredient which isenough for the required or desired therapeutic response, or in otherwords, the amount, which is sufficient to elicit an appreciablebiological response when, administered to a patient. The appreciablebiological response may occur as a result of administration of single ormultiple doses of an active substance. A dose may comprise one or moredosage forms. It will be understood that the specific dose level for anypatient will depend upon a variety of factors including the indicationbeing treated, severity of the indication, patient health, age, gender,weight, diet, pharmacological response, the specific dosage formemployed, and other such factors.

The desired dose for oral administration is up to 5 dosage formsalthough as few as one and as many as ten dosage forms may beadministered as a single dose. Exemplary dosage forms contain 0.1 to 5mg of the SCF extract per dosage form, for a total 0.1 to 500 mg (1 to10 dose levels) per dose. Doses will be administered according to dosingregimens that may be predetermined and/or tailored to achieve specifictherapeutic response or clinical benefit in a subject.

For use in treatment of mammals, the extract, or fraction orsub-fraction thereof, can be included in a dosage form. Some embodimentsof the dosage form are not enteric coated and release their charge ofextract within a period of 0.5 to 1 hours or less. Some embodiments ofthe dosage form are enteric coated and release their charge of cardiacdownstream of the stomach, such as from the jejunum, ileum, smallintestine, and/or large intestine (colon). Enterically coated dosageforms will release the extract into the systemic circulation within 1-10hr after oral administration.

It should be noted that a compound herein might possess one or morefunctions in the formulation of the invention. For example, a compoundmight serve as both a surfactant and a water miscible solvent or as botha surfactant and a water immiscible solvent.

A liquid composition can comprise one or more pharmaceuticallyacceptable liquid carriers. The liquid carrier can be an aqueous,non-aqueous, polar, non-polar, and/or organic carrier. Liquid carriersinclude, by way of example and without limitation, a water misciblesolvent, water immiscible solvent, water, buffer and mixtures thereof.

As used herein, the terms “water soluble solvent” or “water misciblesolvent”, which terms are used interchangeably, refer to an organicliquid which does not form a biphasic mixture with water or issufficiently soluble in water to provide an aqueous solvent mixturecontaining at least five percent of solvent without separation of liquidphases. The solvent is suitable for administration to humans or animals.Exemplary water soluble solvents include, by way of example and withoutlimitation, PEG (poly(ethylene glycol)), PEG 400 (poly(ethylene glycolhaving an approximate molecular weight of about 400), ethanol, acetone,alkanol, alcohol, ether, propylene glycol, glycerin, triacetin,poly(propylene glycol), PVP (poly(vinyl pyrrolidone)),dimethylsulfoxide, N,N-dimethylformamide, formamide,N,N-dimethylacetamide, pyridine, propanol, N-methylacetamide, butanol,soluphor (2-pyrrolidone), pharmasolve (N-methyl-2-pyrrolidone).

As used herein, the terms “water insoluble solvent” or “water immisciblesolvent”, which terms are used interchangeably, refer to an organicliquid which forms a biphasic mixture with water or provides a phaseseparation when the concentration of solvent in water exceeds fivepercent. The solvent is suitable for administration to humans oranimals. Exemplary water insoluble solvents include, by way of exampleand without limitation, medium/long chain triglycerides, oil, castoroil, corn oil, vitamin E, vitamin E derivative, oleic acid, fatty acid,olive oil, softisan 645 (Diglyceryl Caprylate/Caprate/Stearate/Hydroxystearate adipate), miglyol, captex (Captex 350: GlycerylTricaprylate/Caprate/Laurate triglyceride; Captex 355: GlycerylTricaprylate/Caprate triglyceride; Captex 355 EP/NF: GlycerylTricaprylate/Caprate medium chain triglyceride).

Suitable solvents are listed in the “International Conference onHarmonisation of Technical Requirements for Registration ofPharmaceuticals for Human Use (ICH) guidance for industry Q3CImpurities: Residual Solvents” (1997), which makes recommendations as towhat amounts of residual solvents are considered safe inpharmaceuticals. Exemplary solvents are listed as class 2 or class 3solvents. Class 3 solvents include, for example, acetic acid, acetone,anisole, 1-butanol, 2-butanol, butyl acetate, tert-butlymethyl ether,cumene, ethanol, ethyl ether, ethyl acetate, ethyl formate, formic acid,heptane, isobutyl acetate, isopropyl acetate, methyl acetate,methyl-1-butanol, methylethyl ketone, methylisobutyl ketone,2-methyl-1-propanol, pentane, 1-pentanol, 1-propanol, 2-propanol, orpropyl acetate.

Other materials that can be used as water immiscible solvents in theinvention include: Captex 100: Propylene Glycol Dicaprate; Captex 200:Propylene Glycol Dicaprylate/Dicaprate; Captex 200 P: Propylene GlycolDicaprylate/Dicaprate; Propylene Glycol Dicaprylocaprate; Captex 300:Glyceryl Tricaprylate/Caprate; Captex 300 EP/NF: GlycerylTricaprylate/Caprate Medium Chain Triglycerides; Captex 350: GlycerylTricaprylate/Caprate/Laurate; Captex 355: Glyceryl Tricaprylate/Caprate;Captex 355 EP/NF: Glyceryl Tricaprylate/Caprate Medium ChainTriglycerides; Captex 500: Triacetin; Captex 500 P: Triacetin(Pharmaceutical Grade); Captex 800: Propylene Glycol Di(2-Ethythexanoate); Captex 810 D: GlycerylTricaprylate/Caprate/Linoleate; Captex 1000: Glyceryl Tricaprate; CaptexCA: Medium Chain Triglycerides; Captex MCT-170: Medium ChainTriglycerides; Capmul GMO: Glyceryl Monooleate; Capmul GMO-50 EP/NF:Glyceryl Monooleate; Capmul MCM: Medium Chain Mono- & Diglycerides;Capmul MCM C8: Glyceryl Monocaprylate; Capmul MCM C10: GlycerylMonocaprate; Capmul PG-8: Propylene Glycol Monocaprylate; Capmul PG-12:Propylene Glycol Monolaurate; Caprol 10G10O: Decaglycerol Decaoleate;Caprol 3GO: Triglycerol Monooleate; Caprol ET: Polyglycerol Ester ofMixed Fatty Acids; Caprol MPGO: Hexaglycerol Dioleate; Caprol PGE 860:Decaglycerol Mono-, Dioleate.

As used herein, a “surfactant” refers to a compound that comprises polaror charged hydrophilic moieties as well as non-polar hydrophobic(lipophilic) moieties; i.e., a surfactant is amphiphilic. The termsurfactant may refer to one or a mixture of compounds. A surfactant canbe a solubilizing agent, an emulsifying agent or a dispersing agent. Asurfactant can be hydrophilic or hydrophobic.

The hydrophilic surfactant can be any hydrophilic surfactant suitablefor use in pharmaceutical compositions. Such surfactants can be anionic,cationic, zwitterionic or non-ionic, although non-ionic hydrophilicsurfactants are presently preferred. As discussed above, these non-ionichydrophilic surfactants will generally have HLB values greater thanabout 10. Mixtures of hydrophilic surfactants are also within the scopeof the invention.

Similarly, the hydrophobic surfactant can be any hydrophobic surfactantsuitable for use in pharmaceutical compositions. In general, suitablehydrophobic surfactants will have an HLB value less than about 10.Mixtures of hydrophobic surfactants are also within the scope of theinvention.

Examples of additional suitable solubilizer include: alcohols andpolyols, such as ethanol, isopropanol, butanol, benzyl alcohol, ethyleneglycol, propylene glycol, butanediols and isomers thereof, glycerol,pentaerythritol, sorbitol, mannitol, transcutol, dimethyl isosorbide,polyethylene glycol, polypropylene glycol, polyvinylalcohol,hydroxypropyl methylcellulose and other cellulose derivatives,cyclodextrins and cyclodextrin derivatives; ethers of polyethyleneglycols having an average molecular weight of about 200 to about 6000,such as tetrahydrofurfuryl alcohol PEG ether (glycofurol, availablecommercially from BASF under the trade name Tetraglycol) or methoxy PEG(Union Carbide); amides, such as 2-pyrrolidone, 2-piperidone,caprolactam, N-alkylpyrrolidone, N-hydroxyalkylpyrrolidone,N-alkylpiperidone, N-alkylcaprolactam, dimethylacetamide, andpolyvinypyrrolidone; esters, such as ethyl propionate,tributylcitrate,acetyl triethylcitrate, acetyl tributyl citrate,triethylcitrate, ethyl oleate, ethyl caprylate, ethyl butyrate,triacetin, propylene glycol monoacetate, propylene glycol diacetate,caprolactone and isomers thereof, valerolactone and isomers thereof,butyrolactone and isomers thereof; and other solubilizers known in theart, such as dimethyl acetamide, dimethyl isosorbide (Arlasolve DMI(ICI)), N-methyl pyrrolidones (Pharmasolve (ISP)), monooctanoin,diethylene glycol nonoethyl ether (available from Gattefosse under thetrade name Transcutol), and water. Mixtures of solubilizers are alsowithin the scope of the invention.

Except as indicated, compounds mentioned herein are readily availablefrom standard commercial sources.

The clear liquid composition is visually clear to the unaided eye, as itwill contain less than 5%, less than 3% or less than 1% by wt. ofsuspended solids based upon the total weight of the composition.

Although not necessary, a composition or kit of the present inventionmay include a chelating agent, preservative, antioxidant, adsorbents,acidifying agent, alkalizing agent, antifoaming agent, buffering agent,colorant, electrolyte, salt, stabilizer, tonicity modifier, diluent,other pharmaceutical excipient, or a combination thereof.

As used herein, the term “antioxidant” is intended to mean an agent thatinhibits oxidation and is thus used to prevent the deterioration ofpreparations by the oxidative process. Such compounds include, by way ofexample and without limitation, ascorbic acid, ascorbic palmitate,Vitamin E, Vitamin E derivative, butylated hydroxyanisole, butylatedhydroxytoluene, hypophosphorous acid, monothioglycerol, propyl gallate,sodium ascorbate, sodium bisulfite, sodium formaldehyde sulfoxylate,sodium metalbisulfite and other such materials known to those ofordinary skill in the art.

As used herein, the term chelating agent is intended to mean a compoundthat chelates metal ions in solution. Exemplary chelating agents includeEDTA (tetrasodium ethylenediaminetetraacetate), DTPA (pentasodiumdiethylenetriaminepentaacetate), HEDTA (trisodium salt ofN-(hydroxyethyl)-ethylene-diaminetriacetic acid), NTA (trisodiumnitrilotriacetate), disodium ethanoldiglycine (Na₂EDG), sodiumdiethanolglycine (DEGNa), citric acid, and other compounds known tothose of ordinary skill in the art.

As used herein, the term “adsorbent” is intended to mean an agentcapable of holding other molecules onto its surface by physical orchemical (chemisorption) means. Such compounds include, by way ofexample and without limitation, powdered and activated charcoal andother materials known to one of ordinary skill in the art.

As used herein, the term “alkalizing agent” is intended to mean acompound used to provide an alkaline medium. Such compounds include, byway of example and without limitation, ammonia solution, ammoniumcarbonate, diethanolamine, monoethanolamine, potassium hydroxide, sodiumborate, sodium carbonate, sodium bicarbonate, sodium hydroxide,triethanolamine, and trolamine and others known to those of ordinaryskill in the art.

As used herein, the term “acidifying agent” is intended to mean acompound used to provide an acidic medium. Such compounds include, byway of example and without limitation, acetic acid, amino acid, citricacid, fumaric acid and other alpha-hydroxy acids, hydrochloric acid,ascorbic acid, and nitric acid and others known to those of ordinaryskill in the art.

As used herein, the term “antifoaming agent” is intended to mean acompound or compounds that prevents or reduces the amount of foamingthat forms on the surface of the fill composition. Suitable antifoamingagents include by way of example and without limitation, dimethicone,SIMETHICONE, octoxynol and others known to those of ordinary skill inthe art.

As used herein, the term “buffering agent” is intended to mean acompound used to resist a change in pH upon dilution or addition of acidor alkali. Such compounds include, by way of example and withoutlimitation, potassium metaphosphate, potassium phosphate, monobasicsodium acetate and sodium citrate anhydrous and dehydrate and other suchmaterials known to those of ordinary skill in the art.

As used herein, the term “diluent” or “filler” is intended to mean inertsubstances used as fillers to create the desired bulk, flow properties,and compression characteristics in the preparation of tablets andcapsules. Such compounds include, by way of example and withoutlimitation, dibasic calcium phosphate, kaolin, lactose, sucrose,mannitol, microcrystalline cellulose, powdered cellulose, precipitatedcalcium carbonate, sorbitol, and starch and other materials known to oneof ordinary skill in the art.

As used herein, the term “preservative” is intended to mean a compoundused to prevent the growth of microorganisms. Such compounds include, byway of example and without limitation, benzalkonium chloride,benzethonium chloride, benzoic acid, benzyl alcohol, cetylpyridiniumchloride, chlorobutanol, phenol, phenylethyl alcohol, phenylmercuricnitrate, phenylmercuric acetate, thimerosal, metacresol, myristylgammapicolinium chloride, potassium benzoate, potassium sorbate, sodiumbenzoate, sodium propionate, sorbic acid, thymol, and methyl, ethyl,propyl, or butyl parabens and others known to those of ordinary skill inthe art.

As used herein, the term “colorant” is intended to mean a compound usedto impart color to pharmaceutical preparations. Such compounds include,by way of example and without limitation, FD&C Red No. 3, FD&C Red No.20, FD&C Yellow No. 6, FD&C Blue No. 2, FD&C Green No. 5, FD&C OrangeNo. 5, FD&C Red No. 8, caramel, and iron oxide (black, red, yellow),other FD&C dyes and natural coloring agents such as grape skin extract,beet red powder, beta-carotene, annato, carmine, turmeric, paprika,combinations thereof and other such materials known to those of ordinaryskill in the art.

As used herein, the term “stabilizer” is intended to mean a compoundused to stabilize an active agent against physical, chemical, orbiochemical processes that would otherwise reduce the therapeuticactivity of the agent. Suitable stabilizers include, by way of exampleand without limitation, albumin, sialic acid, creatinine, glycine andother amino acids, niacinamide, sodium acetyltryptophonate, zinc oxide,sucrose, glucose, lactose, sorbitol, mannitol, glycerol, polyethyleneglycols, sodium caprylate and sodium saccharin and others known to thoseof ordinary skill in the art.

As used herein, the term “tonicity modifier” is intended to mean acompound or compounds that can be used to adjust the tonicity of theliquid formulation. Suitable tonicity modifiers include glycerin,lactose, mannitol, dextrose, sodium chloride, sodium sulfate, sorbitol,trehalose and others known to those or ordinary skill in the art.

The composition of the invention can also include oils such as fixedoils, peanut oil, sesame oil, cottonseed oil, corn oil and olive oil;fatty acids such as oleic acid, stearic acid and isostearic acid; andfatty acid esters such as ethyl oleate, isopropyl myristate, fatty acidglycerides and acetylated fatty acid glycerides. The composition canalso include alcohol such as ethanol, isopropanol, hexadecyl alcohol,glycerol and propylene glycol; glycerol ketals such as2,2-dimethyl-1,3-dioxolane-4-methanol; ethers such as poly(ethyleneglycol) 450; petroleum hydrocarbons such as mineral oil and petrolatum;water; a pharmaceutically suitable surfactant, suspending agent oremulsifying agent; or mixtures thereof.

It should be understood that the compounds used in the art ofpharmaceutical formulation generally serve a variety of functions orpurposes. Thus, if a compound named herein is mentioned only once or isused to define more than one term herein, its purpose or function shouldnot be construed as being limited solely to that named purpose(s) orfunction(s).

One or more of the components of the formulation can be present in itsfree base or pharmaceutically or analytically acceptable salt form. Asused herein, “pharmaceutically or analytically acceptable salt” refersto a compound that has been modified by reacting it with an acid asneeded to form an ionically bound pair. Examples of acceptable saltsinclude conventional non-toxic salts formed, for example, from non-toxicinorganic or organic acids. Suitable non-toxic salts include thosederived from inorganic acids such as hydrochloric, hydrobromic,sulfuric, sulfonic, sulfamic, phosphoric, nitric and others known tothose of ordinary skill in the art. The salts prepared from organicacids such as amino acids, acetic, propionic, succinic, glycolic,stearic, lactic, malic, tartaric, citric, ascorbic, pamoic, maleic,hydroxymaleic, phenylacetic, glutamic, benzoic, salicylic, sulfanilic,2-acetoxybenzoic, fumaric, toluenesulfonic, methanesulfonic, ethanedisulfonic, oxalic, isethionic, and others known to those of ordinaryskill in the art. Lists of other suitable salts are found in Remington'sPharmaceutical Sciences, 17^(th). ed., Mack Publishing Company, Easton,Pa., 1985, p. 1418, the relevant disclosure of which is herebyincorporated by reference.

The phrase “pharmaceutically acceptable” is employed herein to refer tothose compounds, materials, compositions, and/or dosage forms which are,within the scope of sound medical judgment, suitable for use in contactwith tissues of human beings and animals and without excessive toxicity,irritation, allergic response, or any other problem or complication,commensurate with a reasonable benefit/risk ratio.

A dosage form can be made by any conventional means known in thepharmaceutical industry. A liquid dosage form can be prepared byproviding at least one liquid carrier and oleandrin oroleandrin-containing extract in a container. One or more otherexcipients can be included in the liquid dosage form. A solid dosageform can be prepared by providing at least one solid carrier andoleandrin or oleandrin-containing extract. One or more other excipientscan be included in the solid dosage form.

A dosage form can be packaged using conventional packaging equipment andmaterials. It can be included in a pack, bottle, via, bag, syringe,envelope, packet, blister pack, box, ampoule, or other such container.

The invention includes a method for improving the clinical status of astatistically significant number of subjects of in a population ofsubjects having a neurological condition, the method comprising:administering to the population of subjects an extract of Nerium speciesor Thevetia species, or a composition thereof; and determining theclinical status of the subjects to establish the improved clinicalstatus. In some embodiments, the statistically significant number is atleast 5%, at least 10%, at least 20%, at least 30%, at least 40%, atleast 50%, at least 60%, at least 70%, at least 80% or at least 90% ofthe population. In some embodiments, the extract comprises one or moreother pharmacologically active compounds. In other embodiments, theextract comprises one or more other pharmacologically active compoundscomprises that cooperate with oleandrin or another cardiac glycoside toimprove the clinical status of the subjects.

In view of the above description and the examples below, one of ordinaryskill in the art will be able to practice the invention as claimedwithout undue experimentation. The foregoing will be better understoodwith reference to the following examples that detail certain proceduresfor the preparation of embodiments of the present invention. Allreferences made to these examples are for the purposes of illustration.The following examples should not be considered exhaustive, but merelyillustrative of only a few of the many embodiments contemplated by thepresent invention.

Oleandrin can be purchased from Sigma Chemical Co. (St. Louis, Mo.).

Example 1 Supercritical Fluid Extraction of Powdered Oleander LeavesMethod A. With Carbon Dioxide.

Powdered oleander leaves were prepared by harvesting, washing, anddrying oleander leaf material, then passing the oleander leaf materialthrough a comminuting and dehydrating apparatus such as those describedin U.S. Pat. Nos. 5,236,132, 5,598,979, 6,517,015, and 6,715,705. Theweight of the starting material used was 3.94 kg.

The starting material was combined with pure CO₂ at a pressure of 300bar (30 MPa, 4351 psi) and a temperature of 50° C. (122° F.) in anextractor device. A total of 197 kg of CO₂ was used, to give a solventto raw material ratio of 50:1. The mixture of CO₂ and raw material wasthen passed through a separator device, which changed the pressure andtemperature of the mixture and separated the extract from the carbondioxide.

The extract (65 g) was obtained as a brownish, sticky, viscous materialhaving a nice fragrance. The color was likely caused by chlorophyll. Foran exact yield determination, the tubes and separator were rinsed outwith acetone and the acetone was evaporated to give an addition 9 g ofextract. The total extract amount was 74 g. Based on the weight of thestarting material, the yield of the extract was 1.88%. The content ofoleandrin in the extract was calculated using high pressure liquidchromatography and mass spectrometry to be 560.1 mg, or a yield of0.76%.

Method B. With Mixture of Carbon Dioxide and Ethanol

Powdered oleander leaves were prepared by harvesting, washing, anddrying oleander leaf material, then passing the oleander leaf materialthrough a comminuting and dehydrating apparatus such as those describedin U.S. Pat. Nos. 5,236,132, 5,598,979, 6,517,015, and 6,715,705. Theweight of the starting material used was 3.85 kg.

The starting material was combined with pure CO₂ and 5% ethanol as amodifier at a pressure of 280 bar (28 MPa, 4061 psi) and a temperatureof 50° C. (122° F.) in an extractor device. A total of 160 kg of CO₂ and8 kg ethanol was used, to give a solvent to raw material ratio of 43.6to 1. The mixture of CO₂, ethanol, and raw material was then passedthrough a separator device, which changed the pressure and temperatureof the mixture and separated the extract from the carbon dioxide.

The extract (207 g) was obtained after the removal of ethanol as a darkgreen, sticky, viscous mass obviously containing some chlorophyll. Basedon the weight of the starting material, the yield of the extract was5.38%. The content of oleandrin in the extract was calculated using highpressure liquid chromatography and mass spectrometry to be 1.89 g, or ayield of 2.1%.

Example 2 Hot-Water Extraction of Powdered Oleander Leaves

Hot water extraction is typically used to extract oleandrin and otheractive components from oleander leaves. Examples of hot water extractionprocesses can be found in U.S. Pat. Nos. 5,135,745 and 5,869,060.

A hot water extraction was carried out using 5 g of powdered oleanderleaves. Ten volumes of boiling water (by weight of the oleander startingmaterial) were added to the powdered oleander leaves and the mixture wasstirred constantly for 6 hours. The mixture was then filtered and theleaf residue was collected and extracted again under the sameconditions. The filtrates were combined and lyophilized. The appearanceof the extract was brown. The dried extract material weighed about 1.44g. 34.21 mg of the extract material was dissolved in water and subjectedto oleandrin content analysis using high pressure liquid chromatographyand mass spectrometry. The amount of oleandrin was determined to be 3.68mg. The oleandrin yield, based on the amount of extract, was calculatedto be 0.26%. The table below shows a comparison between the oleandrinyields for the two supercritical carbon dioxide extractions of Example 1and the hot water extraction.

Comparison of Yields Oleandrin yield based Extraction Medium on totalextract weight Supercritical Carbon Dioxide: 0.76% Example 1, Method ASupercritical Carbon Dioxide: 2.1% Example 1, Method B Hot WaterExtraction: Example 2 0.26%

Example 3 Treatment of Neurological Condition Including but not Limitedto Alzheimer's Disease Method A. Extract Therapy

A subject presenting with Alzheimer's disease is prescribed cardiacglycoside, and therapeutically relevant doses are administered to thesubject according to a prescribed dosing regimen for a period of time.The subject's level of therapeutic response is determined periodically.If the level of therapeutic response is too low at one dose, then thedose is escalated according to a predetermined dose escalation scheduleuntil the desired level of therapeutic response in the subject isachieved. Treatment of the subject with the extract, or fraction orsub-fraction thereof, is continued as needed and the dose or dosingregimen can be adjusted as needed until the patient reaches the desiredclinical endpoint.

Method B. Combination Therapy: Extract and Another Therapeutic Agent

Method A, above, is followed except that the subject is prescribed andadministered one or more other therapeutic agents for the treatment ofAlzheimer's disease, or symptoms thereof. Then one or more othertherapeutic agents can be administered before, after or with theextract. Dose escalation (or de-escalation) of the one or more othertherapeutic agents can also be done. Suitable one or more othertherapeutic agents include Namenda™ (memantine HCl), Aricept™(donepezil), Razadyne™ (galantamine), Exelon™ (rivastigmine), Cognex™(tacrine), and amantadine.

Example 4 Treatment of Neurological Condition Including but not Limitedto Huntington's Disease Method A. Extract Therapy

A subject presenting with Huntington's disease is prescribed theextract, and therapeutically relevant doses are administered to thesubject according to a prescribed dosing regimen for a period of time.The subject's level of therapeutic response is determined periodically.If the level of therapeutic response is too low at one dose, then thedose is escalated according to a predetermined dose escalation scheduleuntil the desired level of therapeutic response in the subject isachieved. Treatment of the subject with extract is continued as neededand the dose or dosing regimen can be adjusted as needed until thepatient reaches the desired clinical endpoint. The doses administeredcan be similar to those of Example 3 or as otherwise described herein.

Method B. Combination Therapy: Extract and Another Therapeutic Agent

Method A, above, is followed except that the subject is prescribed andadministered one or more other therapeutic agents for the treatment ofHuntington's disease, or symptoms thereof. The one or more othertherapeutic agents can be administered before, after or with the cardiacglycoside. Dose escalation (or de-escalation) of the one or more othertherapeutic agents can also be done. Suitable one or more othertherapeutic agents include Vitamin E, Baclofen (a derivative of CoQ10),Lamotrigine (an anticonvulsant), remacemide (an anesthetic which is alow affinity NMDA antagonist), and riluzole (Na channel blocker).

Example 5 Treatment of Neurological Condition Including but not Limitedto Ischemic Stroke Method A. Extract Therapy

A subject presenting with ischemic stroke is prescribed the extract, andtherapeutically relevant doses are administered to the subject accordingto a prescribed dosing regimen for a period of time. The subject's levelof therapeutic response is determined periodically. If the level oftherapeutic response is too low at one dose, then the dose is escalatedaccording to a predetermined dose escalation schedule until the desiredlevel of therapeutic response in the subject is achieved. Treatment ofthe subject with the extract is continued as needed and the dose ordosing regimen can be adjusted as needed until the patient reaches thedesired clinical endpoint. The doses administered can be similar tothose in Example 3 or as otherwise described herein.

Method B. Combination Therapy: Extract and Another Therapeutic Agent

Method A, above, is followed except that the subject is prescribed andadministered one or more other therapeutic agents for the treatment ofischemic stroke, or symptoms thereof. The one or more other therapeuticagents can be administered before, after or with the extract. Doseescalation (or de-escalation) of the one or more other therapeuticagents can also be done.

Example 6 HPLC Analysis of Solutions Containing Oleandrin

Samples (oleandrin standard, SCF extract and hot-water extract) wereanalyzed on HPLC (Waters) using the following conditions: Symmetry C18column (5.0 μm, 150×4.6 mm I.D.; Waters); Mobile phase ofMeOH:water=54:46 (v/v) and flow rate at 1.0 ml/min Detection wavelengthwas set at 217 nm. The samples were prepared by dissolving the compoundor extract in a fixed amount of HPLC solvent to achieve an approximatetarget concentration of oleandrin.

Example 7 Determination of α3 and α1 Expression in Normal NeuronalTissue

The procedures set forth in PCT International Application No.PCT/US08/82641, filed Nov. 6, 2008 in the name of Phoenix Biotechnology,Inc., the entire disclosure of which is hereby incorporated byreference, can be followed.

Example 8 Evaluation of a Cardiac Glycoside and an Extract of theInvention in an In Vitro Assay for Stroke and Non-Stroke Method A.Stroke: Preparation of Cortical Brain Slices and OGD.

Neocortical brain slices were prepared from PND 7 Sprague-Dawley ratpups. The cerebral cortex was dissected, cut into 400-μ-thick slices andtransferred into a container containing cold artificial cerebrospinalfluid with 1 μM MK-801 before plating; MK-801 was not included in anysubsequent procedures. To mimic ischemic injury using transientoxygen-glucose deprivation (OGD), slices from one hemisphere of eachbrain were exposed to glucose-free, N₂-bubbled artificial cerebrospinalfluid for 7.5 min in a low O₂ (0.5%) environment. The OGD slices werethen plated side-by-side with control slices from the contralateralhemisphere on nitrocellulose or Millicell (Millipore) permeablemembranes, which were prepared identically except for no OGD. Thirtyminutes after plating, the brain slice pairs were transfected,transferred to 24-well plates, and incubated at 37° C. under 5% CO₂ inhumidified chambers. In each experiment, 5-6 minutes of oxygen-glucosedeprivation (OGD) was used to induce >50% loss of healthy corticalneurons by 24 hrs. A set concentration (3 μM) of neriifolin (a cardiacglycoside) was used as the internal positive control. For oleandrin (acardiac glycoside), all three concentrations from 0.3 to 3 μM appearedto provide neuroprotection in the first two experiments, so theoleandrin concentrations tested were lowered in the third run andsuggested that the threshold concentration for neuroprotection liesbetween 0.1 and 0.3 μM. The unfractionated extract, e.g. of Neriumspecies, or a fraction thereof can also be used as described for theoleandrin.

Method B. Non-Stroke: Brain Slice Assay.

Oleandrin and PBI-05204, an unfractionated SCF extract of Neriumoleander, were tested on “nonstroked” brain slices; that is, ones thatwere sliced and transfected with YFP but not subjected to additionaltrauma via OGD. See experimental procedure outlined above. We haveobserved that a number of neuroprotective compounds, includingneriifolin, can provide modest levels of neuroprotection to such brainslices, presumably by protecting against the trauma caused by theprocess of slicing and culturing itself. The data demonstrate thatoleandrin and the extract appeared to be able to provide neuroprotectionto such “non-OGD” brain slices to similar levels as neriifolinsignifying that cardiac glycosides mediate neuroprotection even in theabsence of oxygen or glucose deprivation.

Example 9 Evaluation of a Cardiac Glycoside and an Extract in an InVitro Assay for Alzheimer's Disease

In the rat brain slice model for APP/Abeta-induced degeneration ofcortical pyramidal neurons biolistic transfection is used not only tointroduce vital markers such as YFP, but also to introduce disease geneconstructs into the same neuronal populations in the brain slices. Thus,the APP/Aβ brain slice model co-transfects YFP with APP isoforms,leading to the progressive degeneration of cortical pyramidal neuronsover the course of 3-4 days after brain slice preparation andtransfection. The data demonstrate that both oleandrin and PBI-05204, anunfractionated SCF extract of Nerium oleander, appeared able to provideconcentration-dependent neuroprotection to APP-transfected brain slices,rescuing to levels nearly to those that can be provided by BACEinhibitor drugs.

Example 10 Evaluation of a Cardiac Glycoside and an Extract in an InVitro Corticostriatal Co-Culture Assay for Huntington's Disease

In this assay, instead of using intact brain slices, mutant htt isintroduced via electroporation into high-density, mixed co-cultures ofcortical neurons, striatal neurons, and glia arrayed in 96-well plates.The goal of this assay platform is to combine the biological/clinicalrelevance of a complex primary culture system that recapitulates keyaspects of the interconnectivity of disease-relevant neuronalpopulations in vivo, with the ability to conduct large-scale fullyautomated screening campaigns. In this assay, over the course of 1-2weeks in vitro, transfected mutant htt constructs induce the progressivedegeneration of both striatal and cortical neurons that are subsequentlyquantified using automated image acquisition and object detectionalgorithms on the Cellomics Arrayscan VTI platform. Each data point wasdrawn from 6 wells with 16 images in each well automatically captured,processed, and analyzed on the Cellomics Arrayscan using protocolsdeveloped during a large-scale screening campaign being conducted inassociation with the Cure Huntington's Disease Initiative. In a fullrun, some 25,000 images are collected and analyzed in each cycle, 4cycles per week.

Cortico-Striatal Co-Culture Assay Platform.

Pure glial cultures are prepared in advance of neuronal plating toestablish 96-well plates with confluent glial beds. Cortical andstriatal tissue are then dissociated separately and “nucleofected” withappropriate DNA constructs and are distinguishable later by theexpression of different fluorescent proteins such as YFP, CFP, andmCherry. These separately transfected cortical and striatal neurons arethen mixed thoroughly and plated into the 96-well plates containing thepreviously plated glial monolayers.

Both oleandrin and PBI-05204 (the supercritical CO₂ extract of Neriumoleander) were tested in this cortico-striatal co-culture platform andpreliminarily these compounds appear to be the strongest hits we haveobserved to date out of >400 late-stage drug molecules that have beenevaluated in this assay system. For comparison, a dose-response graphfor KW6002 (an adenosine 2a receptor antagonist), the compound that weroutinely include as the positive control for this co-culture assay isincluded. Efficacy of oleandrin is on par with KW6002, while its potencyappears to be some 100-fold greater (FIGS. 3A-3D).

Example 11 Evaluation of a Fraction of an SCF Extract of Nerium oleanderin an In Vitro APP Assay for Alzheimer's Disease

The fraction was prepared according to Example 13. This assay wasconducted similar to that of Example 9. The data in FIG. 6 demonstratethat there is a concentration dependent effect of Fraction O-4A inpreventing the neurodegeneration associated with introduction of the APPconstruct. In particular, the data demonstrate neuroprotection betweenthe concentration range of 3 to 30 ug/ml.

Example 12 Evaluation of a Fraction of an SCF Extract of Nerium oleanderin an In Vitro tau4R Assay for Alzheimer's Disease

The fraction was prepared according to Example 13. The data in FIG. 7demonstrate that there is a concentration dependent effect of FractionO-4A in preventing the neurodegeneration associated with introduction ofthe Tau construct. In particular, the data demonstrate a neuroprotectionbetween the concentration range of 3 to 30 ug/ml. There is a significantdifference between Tau construct treated cells and those exposed tosolutions of Fraction O-4A.

Example 13 Chromatographic Fractionation of SCF Extract

A supercritical extract (5 g) of oleander leaves (obtained as describedherein by extracting a plant mass with a mixture of supercritical CO₂with EtOH added as a cosolvent/modifier, Batch #270111) was suspended inwater (150 mL) and partitioned three times with hexane (150 ml eachtime). The water layer was subjected to ODS C-18(octadecyl-functionalized silica gel, 20-22% labeled, 200-400 mesh) opencolumn (400 mm (L)×38 mm (ID)) fractionation by charging the water layerdirectly to a bed of the ODS resin equilibrated with water. The columnwas treated successively with mixtures of water and methanol (1000 ml of30% methanol in water, 1000 ml of 55% methanol in water, 1000 ml of 80%methanol in water, 1000 ml of 100% methanol) and with a mixture ofacetone:methanol (2 volumes: 1 volume; 1000 ml). The effluent (1000 ML)from each mixture was collected. The solvent was removed from eachfraction by evaporation to yield five fractions, namely Fr-O-1, Fr-O-2,Fr-O-3, Fr-O-4, and Fr-O-5. The fractions were then analyzed by HPLCchromatography as per Example 14.

Example 14 HPLC Analysis of Fractions of SCF Extract

The purpose of this assay was to identify extract fractions (from above)containing cardiac glycoside. A sample from each fraction obtainedaccording to Example 13 was analyzed as follows. The fraction 1-3 mg)was dissolved in 1-5 ml of aqueous methanol (80% methanol in water). Thediluted sample (10-25 μl) was analyzed with an Agilent Zorbax SB-C18column using 80% methanol in water as the mobile phase, a flow rate of0.7 mL/min and DAD-UV effluent monitoring at the following wavelengths:203, 210, 217, 230, 254, 280, 310 and 300 nm.

Example 15 Brain-Slice Assay for Determination of NeuroprotectionProvided by Fractions of Extract

This assay was conducted according to Example 8. The data demonstratethat compared to untreated stroke (OGD) mediated damage to brain sliceneurons PBI-05204 provides a significant level of protection. A similarlevel of neuroprotection was provided by Fraction O-4A (FIGS. 4A and 5)as well as Fraction O-3 (FIG. 4C) and Fraction O-1 (FIG. 4D). Incontrast, Fractions O-2 (FIG. 4B) and O-5 (FIG. 4E) demonstrated noneuroprotective effects in this OGD model of stroke mediated ischemicbrain injury.

Example 16 Time-Delay Brain-Slice Assay for Determination ofNeuroprotection

This assay was conducted according to Example 8 except that thefollowing changes were made. A specified length of time was allowedbetween OGD and introduction of a proposed neuroprotective agent. Theability of PBI-05204 to provide neuroprotection to brain slices iftreatment was delayed relative to the timing of the OGD treatment wasdetermined. Data showed that a 2 hr delay of Nerium oleander extractswas well tolerated, showing similar levels of neuroprotection to thoseattained with application of PBI-05204 immediately following OGDtreatment. Neuroprotective benefit was reduced with 4 to 6 hr of delayof administration of PBI-05204, but at levels of neuroprotection thatwere still significantly and physiologically relevant.

Example 17 Identification of Compounds in a Fraction of Nerium oleanderSCF Extract Obtained According to Example 13

The water and methanol present in the Fr-O-4 fraction were removed byevaporation under reduced pressure. The residue from the Fr-O-4 fractionof Example 13 was subjected to silica gel chromatography (below) toprovide sub-fractions that were then analyzed by thin layerchromatography (TLC). Fractions having similar TLC profiles werecombined and the solvents thereof removed by evaporation under reducedpressure. The remaining residues were analyzed by HNMR.

Thin Layer Chromatography

TLC was performed on conventional analytical grade TLC plates using amixture of hexane: ethyl acetate (7:3 v:v). The compounds werevisualized with H₂SO₄, whereby steroids exhibit a blue color andtriterpenes exhibit a purple color.

Prior to further fractionation by flash chromatography, TLC analysis ofthe Fr-O-4 fraction indicated the presence of one major spot and morethan five small spots. The color reaction indicated that the major spotcontained a mixture of steroid and triterpene and most of the smallspots contained steroids.

Silica Gel Flash Chromatography

Silica gel (Biotage; (10-15 g) was loaded into a column and equilibratedwith a mixture of ethyl acetate (3%) and hexane (97%). The residue fromthe Fr-O-4 fraction was taken up in mixture 0.2-0.5 ml] of ethyl acetate(3%) and hexane (97%) and charged onto the column Flash chromatographywas conducted using a solvent gradient of ethyl acetate (3%-30%) inhexane (97%-70%, respectively) followed by 100% methanol. Sub-fractionscollected from the column were analyzed by TLC (above) and thosefractions having similar TLC visualization profiles were combined andconcentrated to remove solvent.

HNMR Spectroscopy

A sample of each of the concentrated sub-fractions obtained from flashchromatography was analyzed by HNMR using conventional methods so as todetermine the structural class for the major components

Example 18 Identification of Compounds in Nerium oleander SCF ExtractObtained According to Example 1 (Method B) in Unfractionated Form

The SCF extract was analyzed by MS-DART TOF analysis as follows. A JEOLAccuTOF-DART mass spectrometer (Jeol U.S.A., Peobody, Mass., U.S.A.) wasused.

A JEOL AccuTOF-DART mass spectrometer (Jeol USA, Peabody, Mass., USA)was used. Analyses were conducted in a positive ion mode (DART+) givingmasses corresponding to the M+H+ ions generated by the DART-MS. A rangeof settings on the instrument was used to determine optimal conditionsfor N. oleander analyses. The general settings for DART+ included:needle voltage 3500 V; orifice 1-2-20 V; ring lens 2-5 V; orifice 2-2-5V; and peaks voltage 1000 V. Calibrations were performed internally witheach sample using a 10% solution of PEG 600 which provides mass markersthroughout the required mass range of 100-1000 mass units. Otheranalyses were undertaken in the DART-mode and these consisted of: needlevoltage 3500 V; heating element 250° C.; electrode 1-150 V; electrode2-250 V; He gas flow rate 3.79 LPM. Mass spectrometer settings: MCP 2600V; orifice 1-15 V; ring lens-5 V, orifice 2-5 V; and peaks voltage 1000V. Calibrations were performed internally with each sample using aperfluorinated carboxylic acid solution that provides markers throughoutthe required mass range of 100-1000 mass units. The N. oleander sampleswere introduced neat into the DART helium plasma using the closed end ofa borosilicate glass melting point tube. The capillary tube was held inthe He plasma for approximately 3-5 s per analysis. Molecular formulaswere confirmed by elemental composition and isotope matching programsprovided with the JEOL AccuTOF DART-MS instrument. A searchable databaseof N. oleander constituents, developed by HerbalScience (Naples, Fla.,USA) was used.

The SCF extract was found to contain at least the following componentspresent in the indicated relative abundances (%).

Component Relative Abundance (%) Oleandrin 2.99 Oleandrigenin 3.31Ursolic acid/betulinic acid 15.29 Odoroside 0.80 Oleanolic acid 0.60Urs-12-ene-3β,28-diol/betulin 5.44 3β,3β-hydroxy-12-olean-en-28-oic acid14.26 28-norurs-12-en-3β-ol 4.94 Urs-12-en-3β-ol 4.76

As used herein and unless otherwise specified, the term “about” or“approximately” are taken to mean±10%, ±5%, ±2.5% or ±1% of a specifiedvalued. As used herein and unless otherwise specified, the term“substantially” is taken to mean “to a large degree”, “at least amajority of”, greater than 70%, greater than 85%, greater than 90%,greater than 95%, greater than 98% or greater than 99%.

The above is a detailed description of particular embodiments of theinvention. It will be appreciated that, although specific embodiments ofthe invention have been described herein for purposes of illustration,various modifications may be made without departing from the spirit andscope of the invention. Accordingly, the invention is not limited exceptas by the appended claims. All of the embodiments disclosed and claimedherein can be made and executed without undue experimentation in lightof the present disclosure.

1) (canceled) 2) (canceled) 3) An extract, or a fraction or sub-fractionthereof of Nerium species or of Thevetia species, the extract, fractionor sub-fraction thereof comprising as major components at least onesteroid and at least one triterpene extracted from the Nerium species orThevetia species, and comprising less than 1% wt. of polysaccharideextracted from the Nerium species or Thevetia species, and excludingoleandrin and neriifolin. 4) A pharmaceutical composition comprising theextract, or fraction or sub-fraction thereof of claim
 3. 5) (canceled)6) (canceled) 7) (canceled) 8) (canceled) 9) (canceled) 10) (canceled)11) (canceled) 12) (canceled) 13) (canceled) 14) (canceled) 15) Theextract, or fraction or sub-fraction thereof of claim 3 furthercomprising one or more therapeutically effective agents extracted fromthe Nerium species or Thevetia species. 16) The extract, or fraction orsub-fraction thereof, of claim 3 further comprising one or more othertherapeutically effective agents. 17) The extract, or fraction orsub-fraction thereof, of claim 3, wherein the extract is obtained byextraction of Nerium species or Thevetia species by hot waterextraction, cold water extraction, supercritical fluid extraction,organic solvent extraction or a combination thereof. 18) The extract, orfraction or sub-fraction thereof, of claim 3, wherein the extractexcludes a therapeutically effective amount of cardiac glycoside. 19)(canceled) 20) The extract, or fraction or sub-fraction thereof, ofclaim 3, wherein the fraction has been prepared by liquidchromatographic fractionation of the extract. 21) The extract, orfraction or sub-fraction thereof of claim 3, wherein the Nerium speciesis Nerium oleander and the Thevetia species is Thevetia neriifolia. 22)The extract, or fraction or sub-fraction thereof of claim 3, wherein thesub-fraction has been prepared by liquid chromatographic fractionationof a fraction of the extract, and the sub-fraction excludes oleandrinand neriifolin. 23) (canceled) 24) A method for preparing a fraction ofextract of Nerium species or Thevetia species comprising: extracting amass comprising Nerium species or Thevetia species to form an extractthereof, the extract comprising one or more pharmacologically activecomponents for the treatment of a neurological condition; andfractionating the extract to form two or more fractions thereof, whereinat least one fraction comprises as major components at least one steroidand at least one triterpene extracted from the Nerium species orThevetia species, comprises less than 1% wt. of polysaccharide extractedfrom the Nerium species or Thevetia species and excludes oleandrin andneriifolin. 25) The method of claim 24, wherein the extracting has beenconducted by hot water extraction, cold water extraction, organicsolvent extraction, supercritical fluid extraction or a combinationthereof. 26) The method of claim 24, wherein the fractionating has beenconducted by liquid chromatography. 27) The method of claim 24, wherein:a) the at least one fraction excludes cardiac glycoside; or b) the atleast one fraction further comprises cardiac glycoside. 28) A method offractionating an extract of Nerium species or Thevetia species in orderto provide one or more therapeutically effective fractions thereof, themethod comprising: a) providing an extract of Nerium species or Thevetiaspecies, wherein the extract comprises at least one triterpene and atleast one steroid and comprises less than 1% wt. of polysaccharideextracted from the Nerium species or Thevetia species; b) fractionatingthe extract to provide two or more different fractions of the extract, afirst extract fraction comprising one or more pharmacologically activeagents, which is/are not a cardiac glycoside, and excluding oleandrinand neriifolin, and a second extract fraction comprising one or morecardiac glycosides and one or more pharmacologically active agents,which is/are not a cardiac glycoside. 29) The method of claim 28,wherein the first or second extract fraction is subjected to furtherfractionation to provide two or more different sub-fractions, wherein afirst sub-fraction comprises one or more steroids and a secondsub-fraction comprises one or more triterpenes. 30) The method of claim28, wherein the fractionation is performed by liquid chromatography witha stationary phase and a mobile phase. 31) A composition comprising asub-fraction of fraction of an extract obtained from Nerium species orThevetia species, whereby the sub-fraction has been obtained by furtherfractionation of a fraction of the extract obtained from Nerium speciesor Thevetia species, and wherein the sub-fraction comprises as majorcomponents at least one steroid and at least one triterpene extractedfrom the Nerium species or Thevetia species, comprises less than 1% wt.of polysaccharide extracted from the Nerium species or Thevetia speciesand excludes oleandrin and neriifolin. 32) The composition of claim 31,wherein: a) the sub-fraction comprises one or more steroids, cardiacglycosides, the associated aglycones of cardiac glycosides,oleandrigenin, cardenolides, or triterpenoids, and one or moretritepenes; b) the sub-fraction comprises one or more triterpenes andexcludes a steroid; and/or c) each sub-fraction independently excludesoleandrin and neriifolin. 33) The extract, or fraction or sub-fractionthereof, of claim 3, wherein the extract comprises at least twopharmacologically active agents obtained (extracted) from Nerium speciesor Thevetia species. 34) The invention according to claim 33, whereinthe at least one steroid and at least one triterpene function additivelyor synergistically to contribute to the therapeutic efficacy of theextract when the extract is administered to a subject. 35) The method ofclaim 28, wherein the cardiac glycoside is selected from the groupconsisting of oleandrin, odoroside, neritaloside, ouabain, bufalin,digitoxin, cinobufatalin, cinobufagin, and resibufogenin. 36) Apharmaceutical composition comprising the composition of claim
 31. 37)The composition of claim 31, wherein: a) the extract has been obtainedfrom an oleander plant mass or Neriifolia plant mass; and b) the plantmass comprises Nerium oleander, Thevetia neriifolia or Thevetiaperuviana. 38) The composition of claim 31, wherein: a) the extract wasprepared by supercritical fluid (SCF) extraction optionally in thepresence of a modifier; b) the extract was prepared by hot waterextraction; c) the extract was prepared by cold water extraction; d) theextract was prepared by organic solvent extraction; e) the extract wasprepared by aqueous organic solvent extraction; or f) the extract wasprepared by a combination of two or more of supercritical fluidextraction, hot water extraction, cold water extraction, or organicsolvent extraction. 39) The extract, or fraction or sub-fraction thereofof claim 3, wherein the extract, or fraction thereof or sub-fractionthereof, of Nerium species or Neriifolia species, has a ¹HNMR spectrumas described any of FIGS. 9B-9I. 40) The extract, or fraction orsub-fraction thereof of claim 3, wherein the extract, or fractionthereof or sub-fraction thereof, of Nerium species or Neriifoliaspecies, exhibits therapeutic activity as described herein whenadministered to a subject. 41) The extract, or fraction or sub-fractionthereof of claim 3, wherein the extract, or fraction thereof orsub-fraction thereof, of Nerium species or Neriifolia species, has aHPLC chromatogram as described in FIG. 8D when chromatographic analysisis conducted as described herein. 42) The extract, or fraction orsub-fraction thereof of claim 3 comprising at least one steroid, atleast two triterpenes and less than 1% wt. of polysaccharide extractedfrom the Nerium species or Thevetia species, and excludes oleandrin andneriifolin. 43) The extract, or fraction or sub-fraction thereof ofclaim 3 comprising at least two steroids, at least two triterpenes andless than 1% wt. of polysaccharide extracted from the Nerium species orThevetia species, and excludes oleandrin and neriifolin. 44) Theextract, or fraction or sub-fraction thereof of claim 3, wherein theextract, fraction thereof or sub-fraction thereof comprises betulin(urs-12-ene-3β,28-diol); 28-norurs-12-en-3β-ol; urs-12-en-3β-ol;3β,3β-hydroxy-12-oleanen-28-oic acid; 3β,20α-dihydroxyurs-21-en-38-oicacid; 3β,27-dihydroxy-12-ursen-38-oic acid;3β,13β-dihydroxyurs-11-en-28-oic acid; 3β,12α-dihydroxyoleanan-28,13β-olide; 3β,27-dihydroxy-12-oleanan-28-oicacid; less than 1% wt. of polysaccharide extracted from the Neriumspecies or Thevetia species, and excludes oleandrin and neriifolin. 45)The extract, or fraction or sub-fraction thereof of claim 3, wherein theextract, fraction thereof or sub-fraction thereof comprisesoleandrigenin, ursolic acid, betulinic acid, odoroside, neritaloside,oleanolic acid and one or more triterpenes and less than 0.5% by weightpolysaccharide extracted from the Nerium species or Thevetia species,and excludes oleandrin and neriifolin.